This futon in the lab lounge is so hard and lumpy I’d’ rather crash on the floor. But it’s nearly as sticky-gray as the table cum journal holder, cum lamp stand at the end of it. I am waiting on some gel tracks to finish. I wearily sit up, grab the ratted copy of PNASty on the coffee-juice soaked table next to the fridge. It comes away from the faux wood-grain surface with a stickysssssss. The journal opens, on cue to, ”Conversion of the chill susceptible fruit fly larva (Drosophila melanogaster) to a freeze-tolerant organism.”

Did I mention I’m also a cryonicist?

My middle name is Drosophila.

Humiliation.

Embarrassment.

Feelings of worthlessness.

Should I call GOD (Grand Old (Mike) Darwin) when I get home? That’s a conversation I can’t have here, or at Starbucks across the street.

GOD knows everything, well, almost everything.

Yeah, I should call him.

He hates it when I call him that, so I guess I should call him Darwin here, or maybe just “him”, when it’s grammatically correct.

I started phoning him after I got turned onto the history of the interaction between scientists and cryonics by something Chris Hayworth wrote.

Then I was pulled into his blog.

This place (where I work) is close to one of the Great Libraries. Periodicals. Films. There’s maybe two places you can go to find out about the history of cryonics and science as it happened: Mike Darwin, and the Library of Congress. When I started, I didn’t know to start with Mike Darwin. I’d have saved a lot of time. But I think it would’ve warped my perspective .

Digging through the stacks of magazines and newspapers from the 1960s and the 1970s, ordering up 35 mm film, kinescopes, and videotapes that were the size of hard drives from 1980′s, was like opening old tombs. That stuff smells. It feels ancient. Dead. Gone.

Darwin is alive. Electric. Now. He ruins the past by making it present.

The gels are done.

I’m done.

Gone.

Home.

The bugdust can wait till tomorrow.

I get Darwin on the Droid and start pouring out my woes about the missed opportunity with frozen flies. He is only mildly moved. “It’s good work,” he says, “not so much because it’s great science, but because it shows people straining to do something, to try, to be clever. I know this will sound impossibly, prickishly arrogant, but this is work that could have been done, and should have been done by a kid in high school, or middle school as a Science Fair project 10, or even 15 years ago. No, no, not the DSC (differential scanning calorimetry), and all the sophisticated science, but the basic work of trying to successfully introduce cryoprotectants into flies, or other larger organisms, and then freeze them successfully. Planaria would be a great model for that!”

“Really?” I replied with some skepticism.

The image of a Justin Bieber, working studiously at my bench, just didn’t crystallize in my mind?

“Hell yes!”

“In the 1970s, students, children, were freezing mammals – reproducing Smith’s work – and Greg Fahy and I had both done experiments with invertebrates (and me with vertebrates) before the Science Fair banned such work. In fact, you can introduce 6% DMSO into gold fish. I never tried to see how much additional ice that lets them tolerate. Now, because all such biological “hacking ” is banned, no kid is going to try things like introducing combinations of molecules like perhaps a membrane protecting sugar such as trehalose, a protective amino acid such as proline, and a small amount of a colligative agent, such as glycerol, DMSO or ethylene glycol into a common pest, like the California garden snail. Can’t be done. They’d send the poor bastard off for a psych referral and counseling. “Tsk, tsk, you maladjusted, mean little bugger,” they’d say. ”Why, the next thing you know, you’ll be pulling the wings off song birds and sniffing your mates’ jockstraps in the locker room.”

“I had to admit, he had a point. ”

“So you’re saying I shouldn’t feel so bad that I didn’t do this experiment 10 years ago?”

“No, I’m saying that as far as your likelihood of brilliant scientific contributions to cryobiology goes, you’re fucked. In my opinion, that window probably closed when you were a graduate student, and it certainly closed after you were a post doc. Any mark you make scientifically now in cryobiology/cryonics will be along the lines of what Donaldson did, and Donaldson was a fucking genius.”

“And I’m guessing you think I’m not?” I replied.

“Who do people always put words in my mouth, and then get royally pissed off at me? I’m glad you’re recording these calls, and I hope you not only save them, but that you actually listen to them some day. Because when you do, you’re going find that, to your considerable surprise, after 20 or 30 years of telling people that “Mike Darwin called you a fucking moron,” in fact, what I really said was nothing at all. Literally, nothing at all. Please, try and remember that.

People have this remarkable tendency to substitute their own dire adjectives at junctures like this when they are forced to confront the hard reality that they are not geniuses, or millionaires, or movies stars, or any other of those nearly impossible ideals and that, at least during this life cycle, they are not going to be. That is one of the most important reasons why we are tangled up in cryonics in the first place! Because, if you stop and think about it even a little, not even George Clooney, or Bill Gates, or Barac Obama, or anybody gets it all. They only get a teeny tiny bit of it: and then they die. Whitney Houston. Fantastic, angelic voice. Beautiful woman. Rich, rich rich! Miserable life. Dead. Great stuff, huh? ” Cryonics isn’t just about any of those things, it’s about all of those things, minus death, and infinitely more, and that’s what makes its transcendent. That’s why the prefix trans keeps popping up spontaneously in cryonics (and everywhere else in human culture).”

“So what do you think I should do?” I ask.

“If you mean what specifically, the answer is, ‘I don’t know.’ And that’s because you are not a PFC and I’m not a general. You’re not a grunt with an IQ of 90, under the authority of a nation-state, that I can order about at my pleasure. If I try to do that, you’ll turn on me like a cornered rat. In fact, odds are, you’ll do that no matter how I choose to interact with you. It’s just that the odds are a lot better that it will happen later, rather than sooner.

So I can’t give you orders. I can’t even really give you specific suggestions, because as soon as I do, you’ll start returning with all kinds of ‘well but’s', because again, it will rapidly degenerate into my planning your life. That won’t work.”

“So what does work?”

“The nature of an insurgency is that, in its early stages, it is self organizing. Still, it must reach a critical mass. How it does that is still a mystery to me. I think it is part chance, part timing, part the presence of the right individual – the nucleating individual.”

“Do you think you’re that nucleator?”

“It doesn’t matter what I think. At any one time there are a thousand, ten thousand, maybe a million guys who think they are the nucleators. I was in the UK at the baths and all the action had stopped. All the men had gathered around the telly to watch this ghastly, absolutely ghastly woman with Asperger’s from Scotland sing.[i] There was no sex to be had anywhere; these men had paid good money to get laid and they’re watching this ghastly woman on TV! She sang. Objectively, her voice was good. Not great, not fantastic. Definitely the kind of voice that can make a meager living for you at the low end of the industry if you have a good personality and a great manager; clearly neither of which she had. Good singing voices are common. Great singing voices, truly great singing voices, are not. Now this, on the telly, commanding the attention of gay men in a city where you can hear the most magnificent voices in the world at St. Martin’s in the Field for fucking free (if you can read)!

As it turned out, she became a sensation, went onto fame in the U.S., sold millions of albums! It was mad, absolutely mad! And I assure you, it had nothing to do with her raw talent. She was one of millions and millions of would-be nucleating agents trying for that peculiar niche, and she was in exactly the right place at exactly the right time. Did she think she was going to be a multimillionaire hit recording star? It doesn’t really matter, because she is. It’s very much like the lottery if you are poor , disenfranchised, have no other options and desperately want to get hold of millions. Well it’s really your only chance, and if you don’t play, you can’t win.

I’d also hasten to add that you’d best be careful what you wish for and be damn sure you have the tools and the talent to handle it if you get it, because most people who win the lottery are destroyed by it. And the results of winning for most insurgents and insurgencies are disastrous for them.”

“But back to me? Where do I fit in?”

“You say you’ve become ‘obsessed’ with the war between the cryobiologists and us. What have you learned?”

“That you single-handedly squashed those Cacks . Reading that history, the history that you wrote of the battle royale between the cryobiologists and the cryonicists, between them and us, I mean, that was the catalyst. When I began looking at the source material, it didn’t compute. ”

“Why not?”

“They caved too quickly. It was all over as if they’d been hit in the taint with a sledge hammer. That didn’t make sense. Cacks don’t wage a 20 year war, invest their reputations and take the time to go on TV and talk to journalists, and then just stop. Not. Doesn’t happen.”

“So?”

“So I wanted to know what did happen. I know that you threatened to sue them. They’re herd animals. But some of them are mavericks. And some of them are stupid, too. ”

“Like Dr. Arthur Rowe, who, in fact, is still alive, and recently, like a frozen Woolly Mammoth in some bad B-movie, has come back to life, eons later, and is making TV appearances again, trashing cryonics.”

“Yeah, like Arthur Rowe.”

“There are colleagues of mine here who won’t talk to any journalist, but if someone from Wired or Scientific American comes sniffing around, they can’t help themselves. Greed and ego, ego and greed.”

“Exactly.”

“So, I wanted to know what happened and that’s when I started digging. I guess that’s when I began to understand your message on Chronosphere and to understand what the word insurgency meant. I think it’s Chris Hayworth who mentions that you threatened to sue the Society for Cryobiology.

When your name comes up in cryonics, everybody thinks they know you, and everyone has a story to tell about you. In a small group of people who’ve been involved for a while, I’m usually the only one that hasn’t got anything to say. Listening to that kind of talk is funny. I sit and think about the letters written to those scientists’ bosses. And to the bosses of those scientists’ bosses. About the phone calls, probably hundreds of phone calls made to university chancellors, blood bank officials, trustee members, university board members, grant committee remembers. About all the letters, hundreds and hundreds of letters on different letter heads, on no letter heads; letters written and mailed to the same types of people complaining about the unscientific, unethical, overreaching and improper behavior of their scientist employees. Courteous letters and not so courteous letters.

And I have to wonder what kinds of letters some of those scientists, or their families, the ones who didn’t stop their unscientific and irrational attacks on cryonics, might have received?”

“I’m sure I wouldn’t know.”

“You know, a few of the secretaries and support staff who worked for some of the most outspoken scientific critics of cryonics are still around. They offer an interesting peek into that time. You ground those people down. In fact, you sacred the crap out of them.”

“I had help.”

“I’m sure you did. But it was you. It was your idea. It was your leadership. It was your insurgency, as you would put it.”

“Yes.”

“Melody Maxim?”

“What about her?”

“She was not merely annoying, she was becoming dangerously destructive. Not because of the true things she was saying. Had she spoke the truth – no matter how malignantly or viciously, no matter with what calls for regulation and policing, I would have remained silent. But she began to lie, to defame good men who were cryopreserved and who could not defend themselves; to threaten the lives of innocent people, and to try to destroy cryonics on the basis of fraud and force. Interestingly, the response of the cryonics organizations (and their members) twenty years after the cryobiologists’ attacks on cryonics organizations that were now orders of magnitude bigger in size and with assets larger still, was to revert to type. It was exactly the same as it had been before 1980. They simply argued with these creeps in their own forums, were picked off one by one, took it, watched the opposition grow dangerously and did nothing. And in the bargain, they fought with each other!

I was stunned. Frankly, I was more stunned than I am today, having just been informed that both my parents have been dead for four months and that I was deliberately not informed about it. It shook me to core. I realized, as I read over that traffic, that cryonics was in no way going to work. It wasn’t an opinion, or a guess, or a hunch, it was a simple fact. It was like turning on the TV on 9/11 and seeing those people falling from one of the Twin Towers. There could be absolutely no question in your mind that whilst those people were alive, they were absolutely certain to be dead within a (short) and quantifiable period of time.

You have to realize that I was not following any of that traffic in real-time. I was busy doing all kinds of other things. In fact, during that immediate time interval, I was in London, soaking up art, music, food, culture and having more sex than any one person should ever have. It was only because of the persistence of this fellow with the handle of Finance Director (FD), who kept intruding into my life to tell me how I was being slandered by this Melody Maxim person, that I even began to read that pap.

And then it took awhile , a long while, to deal with the shock of that “cryonics 9/11.” At least credit me with a lot more sense than George W. Bush. My measured response was to write the “Failure Analysis Lectures” which have been, I must say, a spectacular failure.

But I also began Chronosphere, and I began efforts to squelch the attacks on cryonics. I believe those were successful. Of course, Alcor was also suing Larry Johnson, and I think that that was enormously useful in that it sent the clear message that lies, even if mixed with the truth, will be very costly. They can and will cost you your home, your job, your reputation.

Unfortunately, it is in the nature of the U.S. tort system, and of insurgencies, that they have an inherent dark side. It’s in the nature of any force, of any weapon or technology that there is the capability for harm equal to or greater than that which is present for good. Insurgencies are more like projectile weapons, than, say, bladed weapons, such as knives or swords. As such, they are more suited for warfare and they are mostly of use for killing and mayhem. This is also the difference between the National Guard and the Army, and between the Police and the Army, and it is why you never use the Army in place of the Police. Never. The problem with the Johnson victory is that while most of the book is lies, there is still a meta-truth to it. The “victory”, which was also a shallow one, is thus further diluted, because it was not a completely just one.

There is so little second guessing the fight against the Nazi/Axis ~70 years later because:

the Nazis were kooks,

they behaved with abominable aggressiveness,

their European allies were kooks,

they behaved with disgusting barbarity,

they left the concentration camps to be filmed and photographed,

they were utterly and completely defeated and humiliated,

it was all beautifully documented.

What you witnessed in the ultimate response to Maxim was the rekindling of a mini-insurgency. I gave no orders. Before I came on the scene, Alcor was already prosecuting Johnson, albeit neglecting their flanks with Maxim and Arnold. However, that was not enough then and it is not enough now.

It’s not just about “enemies.” It ‘s about not making progress, about not doing science. It’s about not being excited, planning, thinking, innovating and being obsessed with, and in love with cryonics. The failure to defend ourselves; that’s a symptom of all those other things being absent. Only the sick, the weak, the distracted or the demented fail to defend themselves.”

Yesterday, I learned my parents, both of them, had died a little over 4 months ago. The call came from a staffer at the Alcor Life Extension Foundation. Alcor had been contacted by the attorney handing probate for my parents’ estate. My parents had died within a day of each other. My mother passed on 1 November, my father on 2 November of 2011.

It was not unexpected news. My mother had developed Alzheimer’s disease some years ago and had been frankly demented for the past several years – unable to recognize me or hold meaningful conversations for the past two years. About 8 months ago, my father, 90 years old, informed me, during one of our increasingly infrequent and unpleasant phone calls, that he was not going to call me when my mother died. My response was to inform him that I had no plans for further phone calls to him. It was the end of what had been a sharply deteriorating relationship since my mother’s illness eliminated her role as a buffer between us – a role I had not even understood existed, let alone previously appreciated was necessary.

I had no bad blood with my few other remaining relatives in Indiana, but they apparently chose not to notify me, either. To be fair, I found it difficult to communicate with them and I’m sure the same was true for them. Neither of our phones or mailboxes were often, dare I say ever, burdened with communications.

Mike Darwin and his parents, April, 1955

My parents lived long, happy and productive lives. They gave me a great childhood, free of cares and worries, and afforded me every opportunity for education, knowledge and personal growth. My youth was a time of warmth and loving security. My parents worked hard, earned and enjoyed financial security, and enjoyed a long and happy retirement; free from worry or want. Their “golden years” were spent in remarkably good health. My father, despite being a 3-pack a day smoker since age 13, was lucky to escape with only an aortic replacement, a carotid endarterectomy and a coronary angioplasty, all of which he made rapid and astonishingly complete recoveries from. Aside from a few months of morbidity associated with these illnesses, his retirement years were active and free from any significant cognitive impairment. My mother also remained active and cognitively functional into her late 80s. Both my parents enjoyed active social lives diminished only by the relentless and ever accelerating loss of dear friends, most of whom they had the good (or mis-) fortune to outlive, depending upon your point of view. By the time they reached their mid-80s, they had outlived almost all of their cohorts. This took an especially heavy toll on my mother, who defined herself to a far greater extent than did my father, through her social relationships and through her shared memories with her girlhood friends.

One of the many backyard social gatherings with friends and neighbors my parents held. My mothers is the lady in the big sunglasses. Photo is circa mid-to early 1970s.

How many parents would let their 13 year old kid freeze a veritable zoo of animals, or send turtles careening off into the stratosphere? And how many loving parents (and they were loving parents) would their 14 year old son go off to spend summers with a mad body freezer on Long Island, and, a scant 3 years later, run off to “freeze dead bodies” in the same place – and take a week of his senior year in high school to do so in the bargain?

Me at the Cryonics Society of New York in the summer of 1972.

Me freezing “dead” people in 1973 at age 17.

My parents gave me a great childhood. They offered me every opportunity for education and personal growth any boy could want and as only child they and I had the economic opportunity for both toys in an abundance that many children in larger families don’t enjoy. I’d like to think that both they and I took full advantage of that opportunity.

Clockwise: Christmas, 1956, Halloweenwith my dad, 1957, a von Braun rocket set with “grandma” looking on circa 1962, playing with rabbit in the early 1960s, summer in New York city in 1962.

In looking over the hundreds of photos that now constitute almost all that is left of my parents’ past, I am struck by the evidence therein, or rather lack of evidence, of my integration into their lives after the onset of puberty. This reflects the deep sense of alienation that I felt, as well the visible absence reflected in the photographic record. Not only was I was sexually alienated from the lives they were leading by the biological accident of being homosexual – I was morally and intellectually alienated, as well. For it was at this time that I realized that religion was a farce, that death was both a great evil and personally unacceptable, and that the social and moral constructs on which the civilization I was embedded in were based were, at best, a pastiche of make believe and brutal pragmatism held together with spit and sealing wax.

Thus, intellectually, I had very little attachment to my parents. And as time went on, that meant that increasingly I had less and less emotional attachment, as well. Being home with them for visits was awkward under the best of circumstances, and had been for many years. Gratitude isn’t the same thing as genuine intimacy. My mother’s love and longing for me – the me she remembered – was tragic and pitiful – in large measure because it could not be returned – that person had long ago ceased to exist – and there was no possibility of the easy, spontaneous interaction that been there as a child. In its place was a forced simulacrum that had to be called up mechanically.

And then, she ceased to exist – which was both terrible and terrifying.

When I spoke with the probate attorney’s secretary, I was also not surprised to find that my father had replaced me as the executor and the beneficiary of the estate. My parents loathed cryonics. That is why, in no small measure, I have such high praise for them as parents for in allowing me the autonomy they did, and especially at such a young age to pursue it (cryonics). My mother, in particular, was continually nervous that I was going to “freeze her” and in fact, during her last days “semi-compos mente” whist hospitalized and gravely ill, she grasped my hand and earnestly pleaded with me, “not freeze me – or my brain!” What goes around comes around, and I had far too much love and respect for the autonomy they had shown me, so many years before, and at such a high emotional cost to themselves, to betray them in that way. They should have had no worries – and they should have known that that was the case.

My mother clearly loved me very much and she showed that in countless ways, small and large over the years, right up until she became demented. However, from the time I left Indianapolis in 1981, my parents never came to visit me in California, nor did they call me more than once or twice. When the Alcor facility opened in Riverside, I pleaded with them to come to the Grand Opening. They declined. They came to Las Vegas several times to vacation and they visited friends and family elsewhere on the West Coast – but never me. I never asked them to accept or to believe in cryonics, let alone my homosexuality. But I did ask them to accept a moment of what I considered genuine triumph in my life – the building of Alcor into a respectable place and organization that was not a seedy back-room garage operation. All they had to do was to show up – they could even have come afterwards, and just walked through the place. That rejection was incredibly wounding and, unlike my sexuality, it was not necessary and it was not rooted in religion or morality. Later, with the success of 21st Century Medicine I had another triumph, the successful recovery of dogs with no neurological deficit after 15+ minutes of complete cardiac arrest at 37°C. Again, I asked that they come. Again, they refused. That time, cryonics was not at issue. For me, that was, I think (in hindsight) the final divide between me and them, between ‘us’ and ‘them.’ It was then that I realized that symmetry. Just as I had, many years before as a boy becoming man, felt alienated from and unable to participate in their lives and in their world, so too had they been alienated from and unable to participate in mine. At last, the circle was complete. As I remarked to a dear friend later: “I’m not sure about us cryonicists and the rest of the world. Are they ants that gave birth to giants, or are we giants that gave birth to ants?” His, answer was as true as it was wise: “Both.”

Over the subsequent years, and especially after the full maturation of my bipolar disorder and my breakdown in early 2003, my father became increasingly venomous about cryonics and about me, losing no chance to denigrate or deride either of us – pointing out that I was an abject failure, an impoverished “nut case” that his tax dollars were supporting; and that if my mother had anything to say about it, his money would probably keep supporting me after he was dead – and most likely even after I was dead. I suppose there is truth in what he said. But it was very wounding.

However, the ultimate truth, which I remain convinced of, is that he was wrong about cryonics. Certainly, he was wrong about his money supporting me, either after his death, or mine.That was a simple matter his own actions quite simply, and quite righteously saw to.

The day after I got the news about my parents death, Dr. Brian Wowk kindly offered his condolences and in so doing he used the term “disinherited.” That shocked me, because I in no way feel (or felt) disinherited. This so because I never considered my parents’ money mine. I told them this often, and for many, many years when they were alive. Starting from when I was a teenager, actually. I didn’t earn that money – they did. I told them to spend it on themselves. And as they lived into old age in good health, I cautioned them to save for “spend down” and for the quality nursing home and assisted living care they would very likely need. As it was, they both had and were able to pay for very good nursing home and assisted living care until the day they died. I never wanted nor expected their money. So, I suffered no hurt at all about being “disinherited.” If my father wanted the money to go elsewhere, then I’m happy he was able to see, or at least know, it would do so.

One of the things my parents had no way of knowing I would learn as a teenager banging around the Cryonics Society of New York (CSNY),was the utter contempt I would learn for inheritance – for the very concept of it – and for its fundamental incompatibility with a cryonicist/immortalist existence. My days as a kid at CSNY made me sick to the core at the avarice of children for the unearned money of their dead parents. Seeing that contemptible greed in action sickened me on inheritances at an early age; and nothing in my subsequent experience – right on through to fantastic grab for the wealth of Dick Jones, did anything to improve my opinion of it. I still wince every time I think of, or look at a picture of Clara Dostal – and that is often, since one of she and I hangs on the wall next to where I am sitting now, as I write this. Inheritance is based on the FACT of and the INEVITABILITY of death. And that fact is anathema to us. It is also based on the concept of the unearned at the expense of the lives of the others. And that concept ought to be anathema to everyone.

No, the only things that distresses me about the way my parents passing was handled were that I wasn’t told about their deaths until four month later, and about the obituary my father prepared for submission to the local paper. I would be dishonest if I said I was not relieved about being freed from the socially expected obligations, (and the attendant financial and psychological/emotional ones), of attending the funeral/burial. I said my goodbyes to my mom several years ago, when she was still barely oriented enough to understand. Burials and funeral Masses are rituals for them, not us. They are things for us only when we fail. When they are things of conscious choice made by others, they are unnecessary horrors, and we are under no obligation to participate.

As long as I live, I will not forget my parents, nor will I ever cease to be grateful to them. But they chose, quite consciously, to die. I respected their right to that decision and to their autonomy in making it. But it is a terrible and forever isolating thing to do. It is a thing that starts isolating and alienating years before death actually occurs, because once you accept death and decide to die, you must, inevitably, begin surrendering the struggle to stay involved with life and living, and thus to stay current and a part of the world of today.

This was something that both of them did increasingly, quite independent of their involuntary, age-associated deteriorating cognitive reserves. And that is one huge difference I’m increasingly noticing with experience between cryonicists and non-cryonicists. Even those cryonicists who are sorely neurocognitively challenged struggle mightily to stay involved with, and in love with life and the technologies that drive it. Men like Curtis Henderson and Bob Krueger come to mind. I am humbled and in awe of the nobility of their struggles, and of their courage in confronting the debilities of old age.

I would never call my parents cowards, but there is something terrible, small and lacking in their resignation to death and in their lack of vision. They are in a graveyard now, side by side. It is for that, and for their very conscious choice to be there, that I grieve for them.

No doubt much of the pain I am now feeling is socially programmed. Some of it is genuine sorrow at the loss of what was and what can never be again – brought to the forefront of consciousness by the reality of their deaths. Some of it is, no doubt, the realization of the loathing that my father had for me – a loathing so great that he chose not to even acknowledge me as his son in the obituary he prepared for the mortuary to submit to the local paper.

Ella and Michael Federowicz

Ella A. Federowicz

Michael B. Federowicz

Ella A. Federowicz, 90, Indianapolis, passed away Tuesday November 1, 2011 and her husband Michael B. Federowicz, 90, Indianapolis, passed away Wednesday November 2, 2011. Ella was born in Indianapolis on August 6, 1921 to William and Carrie Forway Rohrman. She retired in 1981 as the supervisor of data entry from Dow Chemical after working there for 25 years. Michael was born in Brooklyn, New York on January 1, 1921 to Benjamin and Constance Jakuc Federowicz. He retired from the Indianapolis Police Department with the rank of Sergeant in 1985 after 31 years of service. Michael also served in the U.S. Army for over 10 years during WWII and the Korean War. He was a member of the Knights of Columbus Council 3660, Fraternal Order of Police Lodge 86, IPD Retired Officers and the Ernie Pyle Post VFW. Ella was preceded in death by her brothers, Virgil and Irvin Rohrman and Michael was preceded by his sister, Anna Kraska. They are survived by a sister-in-law, Janis Rohrman and several nieces and nephews.

A Mass of Christian Burial will be celebrated for Ella and Michael on Tuesday November 8, 2011 at 11 a.m. at St. Barnabas Catholic Church where they were members. Visitation will be Tuesday from 10 a.m. until 11 a.m. at the church. Burial will be in Calvary Cemetery. Online condolences may be shared at: orileyfuneralhome.com

Predicting the future is a tough business. It is an especially tough business when it is proposed that the prediction be highly specific and technically accurate. Say, akin to predicting the iPhone with Siri in 1965. It’s often been noted that none of the Golden Age of Science Fiction writers like Heinlein, Clarke, or Asimov predicted the PC, let alone the laptop. And most didn’t have a clue about the emerging developments in biology. So, the odds that one of those esteemed gentlemen would have conjured up a hand-held device that you could ask just about any question to (and get a useful answer), pay your bills through, order your meals with, get directions from, do your banking over, get reminders, entertainment or voice mail from and have a conversation with…well, the odds of that were just about nil. Just about, but not, as it turns out, quite nil.

In his 1965 cryonics novel, The Age Of The Pussyfoot, that Golden Age Science fiction writer, co-contemporary and friend of Bob Ettinger, Fred Pohl posited the existence of a device called the Joymaker, which every civilized person would necessarily have to have. The Joymaker incorporated the following features and uses:

The Joymaker offers voice mail which is the core of interpersonal interaction in the novel.

Orders all food and beverages and arranges payment, both in the home and in public.

Orders all other goods for delivery and since payment is automatic, the expense of items is not always apparent to the buyers. Thus, the protagonist rapidly depletes his “fortune.”

Replaces the public address system allowing any group of people to hear a public announcement on their Joymakers thus eliminating the need for loudspeakers in public places or interruption of entertainment programming.

Locating people. The central computer can track the position of any Joymaker, and by extension, its owner. This information can be made available at the owner’s discretion.

Jobs not requiring physical presence. One character is a “Reacter,” someone who samples new products and reports her reactions using the Joymaker. The central computer analyzes her reactions in the light of her known psychological makeup and is able to statistically predict how well the product will sell.

Left: Robert C. W. Ettinger, the father of cryonics.

The Age Of The Pussyfoot was set in the year 2527. However, in his Afterword to the novel, Pohl noted that he thought many of the functions of the Joymaker would be realized not in five centuries, but more likely in five decades. Forty seven years after Pussyfoot, the iPhone with Siri is here, and most of Pohl’s predictions are indeed a reality. And, at age 93, Fred Pohl has survived long enough to see his predictions become reality. His friend and fellow science fiction writer Bob Ettinger was cryopreserved late last year and Pohl has been intimately aware of cryonics for ~50 years. He was one of the first people Ettinger contacted about the idea and over the ensuing five decades Ettinger never ceased to nag Pohl to make cryonics arrangements. The two were good friends and stayed in touch in writing – the last letter Ettinger wrote to Pohl shortly before his cryopreservation, admonished him, yet again, to get signed up for cryonics.

I too had tried to persuade Pohl to make cryonics arrangements, even offering him a “free freeze” in 1978. When Ettinger entered cryopreservation on July 23, 2011, Pohl wrote a moving tribute him on his blog “The way of the Future” and this prompted me to take up where Bob necessarily left off in urging Fred to make cryonics arrangements:

Mike Darwinsays: Hello, Fred, this is from Mike Darwin, the guy who made you the offer of a “free freeze” after dinner that night in Louisville, KY in our suite in the Galt House hotel. You were the Guest of Honor at the American Science Fiction Convention in 1978, and we took you to dinner and made you an offer that, as it turned out, you easily could refuse! If you want to read an account of that meeting from the perspective of the cryonics people present at that time, it’s up on line, here: http://www.alcor.org/cryonics/cryonics8301.txt and is entitled, “When You Can’t Even Give it Away – Cryonics and Fred Pohl.

When you write about Bob Ettinger, “He wrote me one more letter, good-naturedly urging me to change my mind. That was the end,” I would say in response, “Uh, uh, it is much more likely, on the basis of probability alone, that was the end not for Bob, but for you.

Bob and I talked and corresponded about you a number of times over the years. Unlike you, I was not close to Bob, and we were often at odds. Interestingly, one of the few things that ever resulted in a genuine emotional connection between us was the offer we made to cryopreserve you for free. While he was too reserved and diplomatic to say so, your given reason for turning cryonics down, well, to be frank, I think it pissed him off a little. It was apparent that he genuinely liked and admired you and that, maybe just as importantly, he shared a common past with you. You and he grew up in the Golden Age of Science Fiction and you both shared the common narrative and heritage of what is now being called “The Greatest Generation.” The last time I saw Bob, was over dinner a few years ago in Michigan. He was quite frail, but wickedly lucid. I asked him if you were still compos mente and if he was still in touch with you. He sighed, “Yes,” and a “Yes.” And then he momentarily lost his temper, which is something I almost never saw him do. I don’t remember his exact words, but they were pretty to close to this: “I guess he doesn’t think that much of me or of the rest us, because he’s so worried about being alone and displaced from the people he knows and loves now. Doesn’t he think I’ll be there? Doesn’t he think any of the hundred or so others from our generation will be there? And if he does, and he is so worried about loneliness and social isolation, then dammit why doesn’t he come along to keep us company?”

I thought that was an extraordinarily good question. But logical and emotional arguments aside, it was painfully clear to me that HE WANTED YOU ALONG FOR THE RIDE. I had a hard time holding back the tears, and I had to excuse myself to the men’s room.

When most men die, their probability for any future goes to zero; in effect, their event horizon collapses. That’s about to happen to you (and to me, and to everyone else). Say what you will, Bob Ettinger now confronts two possibilities – oblivion, or one hell of a really interesting future. A future far more fantastic than anything you or he ever dreamed of, or wrote about. If nothing else, just to have come that far and to be in that position, well, it’s a hell of an accomplishment. And I am very grateful to Bob Ettinger for achieving it, because it opens that possibility to me, as well.

So, Fred, here’s the deal. Your friend is waiting for you: he damn sure wanted you to embark on the adventure (good or bad) that he has now begun. In fact, he kept at you to go until, literally, almost his last breath for this life cycle. He can’t do it anymore, so I guess it is my turn, once again, to ask you to reconsider and to join your friend and colleague on his journey into the land you both dreamed of when you were young, and in your salad days. Please, reconsider your arguments. It is now for sure you won’t be without a friend and cohort, and I can pretty much guarantee you that your revival won’t take place unless you have a use.
Finally, I can tell you for a fact that the best use you have is continue living and growing and telling stories. At our core, we humans are ‘store creatures,’ and we will remain so as long we *are* human. It goes without saying that story creatures need storytellers; your job is thus secure.

Mike Darwin’s response to my piece on the loss of that very good man, Bob Ettinger, caught me completely unaware. I am grateful to you for repeating the offer of a free freeze, Mike, just as I am grateful to the people who sometimes tell me that they’re going to pray for me. Even though I can’t accept your offer, it’s a kind thought.

Let me quote from a poem that was written long ago by John Dryden, in an attempt to sum up the teachings on this subject of the even longer ago Roman philosopher Lucretius. The last six lines say it all, but I’ll give you the whole thing. It goes like this:

So, when our mortal forms shall be disjoin’d.
The lifeless lump uncoupled from the mind,
From sense of grief and pain we shall be free,
We shall not feel, because we shall not be.

Though earth in seas, and seas in heaven were lost
We should not move, we should only be toss’d.
Nay, e’en suppose when we have suffer’d fate
The soul should feel in her divided state,
What’s that to us? For we are only we
While souls and bodies in one frame agree.

Nay, though our atoms should revolve by chance,
And matter leap into the former dance,
Though time our life and motion should restore.
And make our bodies what they were before,
What gain to us would all this bustle bring?
The new-made man would be another thing.

But I do appreciate the offer.

This entry was posted on September 9, 2011 at 12:30 am at http://www.thewaythefutureblogs.com/2011/09/declining-immortality-twice/

Fred Pohl may be the first man in the history of the world to have declined a shot at immortality not once, but twice! I would argue that the really amazing thing about Pussyfoot is not just that Pohl got the technology of the Joymaker right, but that he also got the biotechnology of the future more or less right – granted in no small measure due to that “good man” and good friend of his, Bob Ettinger. Fred Pohl knew a sound and reasonable idea when he saw one , biological or otherwise, and 50 years later cryonics has endured and the biological basis for it has grown steadily better. Lucky patients cryopreserved with little or no ischemia, using the best available vitrification techniques today, will have intact connectomes and minimal neuronal molecular damage. Such fortunate patients will suffer virtually no freezing damage.

Above: The Connectome.

Any yet, Pohl is having none of it.

Right: Viktor Frankel.

I used to find this a mystery. To be surprised by it. To marvel at it. However, that time has long past. The first insight that offered a partial answer to that mystery came from Viktor Frankel’s book, Man’s Search for Meaning. Frankel noted that there were two basic types of people in the concentration camps – those who drew their sense of identity and purpose from their social/societal position; husband, father, lawyer, doctor, mother, grandmother… and those who drew it from some other source, independent of their social context, or how they were labeled. For some, the origin of that sense of identity was religious, for others, it existed independent of any institutional or religious thoughts or beliefs. Those few people saw themselves as unique and worthwhile individuals deserving of and entitled to life and survival at all costs, independent of any external factors or forces.

Much later I realized that another component in the will to survive that is often material in making the choice for cryonics is the yearning to be transcendent. It is not enough to be able to see the future with accuracy and precision, it is necessary to yearn to be it. To quote Nietzsche:

”I teach you the overman. Man is something that shall be overcome. What have you done to overcome him? … All beings so far have created something beyond themselves; and do you want to be the ebb of this great flood, and even go back to the beasts rather than overcome man? What is ape to man? A laughing stock or painful embarrassment. And man shall be that to overman: a laughingstock or painful embarrassment. You have made your way from worm to man, and much in you is still worm. Once you were apes, and even now, too, man is more ape than any ape…. The overman is the meaning of the earth. Let your will say: the overman shall be the meaning of the earth…. Man is a rope, tied between beast and overman—a rope over an abyss … what is great in man is that he is a bridge and not an end.”

H. G. Wells said it far more beautifully:

“We look back through countless millions of years and see the great will to live struggling out of the intertidal slime, struggling from shape to shape and from power to power, crawling and then walking confidently upon the land, struggling generation after generation to master the air, creeping down the darkness of the deep; we see it turn upon itself in rage and hunger and reshape itself anew, we watch it draw nearer and more akin to us, expanding, elaborating itself, pursuing its relentless inconceivable purpose, until at last it reaches us and its being beats through our brains and arteries…It is possible to believe that all the past is but the beginning of a beginning, and that all that is and has been is but the twilight of the dawn. It is possible to believe that all that the human mind has accomplished is but the dream before the awakening; out of our lineage, minds will spring that will reach back to us in our littleness to know us better than we know ourselves. A day will come, one day in the unending succession of days, when beings, beings who are now latent in our thoughts and hidden in our loins, shall stand upon this earth as one stands upon a footstool, and shall laugh and reach out their hands amidst the stars.”

But Wells spoke of not of achieving that greatness personally, but rather of the species achieving it – of our descendants achieving it.

To want it, to need it, to ache for it personally – that is a rare thing. It is the motive force that has driven biological evolution – and it is the motive force that has driven every human innovation and every human conquest – for good or evil.

Recently, a friend of mine asked, in wonder, why I was preparing for the contingency that technological civilization might collapse. “There would be no cryonics if that happened.” he noted, correctly.

“Yes, I know.” I replied.

“And it would be really horrible. A terrible, terrible undoing of the world.” he added.

“Yes, yes it would.” I agreed.

“Then why on earth would you want to be around to see that?”

“I can’t imagine missing the last act! I mean, honestly, I’ve had the chance to read up on all that happened before, I’ve trotted all over the planet, read the thoughts of the best minds of every known culture and civilization, and you propose I should wimp out and miss the denouement? I’m plenty savvy enough to keep redundant assets for a quick and painless exit at should I find myself in unbearable agony and no hope of survival. However, absent that, I can’t even conceive of betraying the intense curiosity I’d have about any apocalypse, even if my own survival were impossible.”

Frankel comes close to summing up my feelings on this matter when he says: ”Man is that being who invented the gas chambers of Auschwitz; however, he is also that being who entered those gas chambers upright, with the Lord’s Prayer or the Shema Yisrael on his lips.” There is an implied qualification not present in Frankel’s quote: “Man at his best is that…” The cryonicist is thus that being who chooses life, inquiry, knowledge and understanding of the universe as his personal and moral imperatives. He chooses to feel and to be these things – not just to think about them, or talk about them. He chooses action over contemplation, life over death.

The origins of that choice? Well, that is still a mystery, but one which, in the fullness of time, may we may hope to unravel.

I think every cryonicist carries in his head his own unique model of the “future of cryonics.” Furthermore, I think that each individual cryonicist carries around a largely arbitrary and unique set of standards, rules and regulations concerning what constitutes “proper,” “moral,” “ethical,” or even “reasonable” behavior for both “rank and file” and “professional” cryonicists.

We cryonicists often use the words “movement” or “industry” to describe our undertaking. However, it is a commonplace to all real movements, industries (and, I would add professions) that they share at least a broad world view and a basic, common vision of the future; as well a reasonably well developed set of rules, regulations, guidelines and ethics for carrying out day to day operations. A corollary of such a basic self-regulatory framework is a “judiciary” to enforce these obligations and injunctions.

Medicine, the law, other professions, and even academia, the trades and trade unions have such value-driven enforcement mechanisms in place. In all these examples, senior and respected members of the profession, trade, or ideological movement[i] serve as appointed adjudicators to both fairly and responsibly enforce both the objective and subjective codes of behavior that have been put in place over time.

In the case of medicine, there are both private, professional organizations and state-sponsored, or state-informed organizations, such as the state medical boards, whose job it is to set and enforce a minimum standard of “right” conduct, which is understood to include moral, ethical and legal behavior. These entities do not function in a “black or white,” “all or none,” “guilty or innocent” manner. Rather, they consider the totality of the cases that come before them and attempt to reach a just resolution. For instance, under most conditions, it is unethical for a physician to engage in sexual congress with a patient. However, depending upon the circumstances, including the prior professional history of the physician, such a transgression may be handled by a simple reprimand, or alternatively, by being struck out of the profession for life.

In any mature ideological movement, religious, political, social, or otherwise, there are similar “rules and regulations” and a well defined world view and vision of the future. There may well be (and usually are) both conservatives and radicals in any given organization with respect to this world view and vision (and usually many more who are “moderates”). However, this does not prevent or preclude there being clearly and objectively stated rules. There are members of the American Medical Association who support active euthanasia and more than a few Roman Catholics who support (and use) birth control.

What does this have to do with the “future of cryonics”? Quite a lot, really, because the expectations of members and leaders within cryonics organizations will shape the actions taken by the cryonics organization as a whole – even if that “shaping” is to effectively preclude coordinated action.

For instance, cryonicists who envision rapid and largely unimpeded technological progress sufficient to make cryonics “successful” (i.e., to achieve perfected suspended animation, or to resuscitate today’s cryopatients) will likely find conflict in the brass tacks of dealing with cryonicists who have a contrary view of the future – who see the future as a difficult and dangerous place and believe that cryonics must largely make its own way and forge its own advances – and if necessary, alter the course and values of the global culture to facilitate the survival of cryonicists (both living and cryopreserved).

It is also the case that any enterprise operating completely sans written minimum standards, rules, regulations, obligations and moral and ethical expectations for its leadership and its membership will function chaotically and ultimately, will fail.

This is most evident in the case of Alcor, where the proof of such a standard-less or “lawless” operation can be found in the high turnover of management and staff.[ii] Some years ago, I was riding in a car with then Alcor President Steven Van Sickle. He remarked that he wanted to have T-shirts made up for all the Alcor Presidents, past and present, with a bull’s eye printed on them along with words to the effect of “shoot here” and “invite them to attend the next Alcor Conference to wear the shirts.” The sentiment he was expressing was that no matter what you do, you will eventually be found summarily guilty and shot. That is a true and sure sign of an organization without standards that lurches from decision to decision based on the expediency of the moment, whether it be cash flow, number of cryopreservations per year, membership growth, or avoiding a “catastrophe” of one sort or another (justified or unjustified).

Even where there are standards that are well known and written down (somewhere), such as the conditions under which at-need cases should be accepted, they are violated (as in the case of Ted Williams) – usually because those making the decisions had no mentoring and no inculturation in such rules. People do not learn “right” or “proper” behavior by being once “told the rules,” or by being given a stack of papers where they are written down (or engraved on stone, for that matter); any more than they learn moral or ethical behavior in daily life in that fashion. Such behavior, and the values that underlie them, are inculcated more than educated. No one even learns the mechanics of driving a car from reading the state-provided operator’s manual – sans lots of practice andmentoring during the actual business of operating a motor vehicle. And this is doubly true for the large body of mostly unwritten behavior that constitutes being a courteous driver! That can only be acquired from mentoring and from repetition and observation of the good conduct of others who are vastly more experienced, and for whom there is genuine respect.

The fatal flaw in Ettinger’s vision of cryonics was that cryonics itself was to come from them and not from us. In his world view, large corporations and the government would become involved almost from the beginning, as well as the trades and professions, and they would then work out all the details of what constituted right and proper conduct in every sphere of action – from rescue – through storage and “reanimation.” Clearly, that didn’t happen. And, by the way, there was no great sin in imagining that it might. The world is a wild, crazy and unpredictable place and it seems eminently possible to me that in some universe somewhere there is indeed the “freezer centered society” that Ettinger envisioned in 1962-4.

Rather, the sin is that 50+ years later, we still have not awakened to the reality that this culture and this civilization are, at best, monumentally indifferent to our undertaking and worst in deadly opposition to it.

We have a profound responsibility to arrive at a world view, a morality and code of conduct of for cryonics. That these should be reasonably inclusive and flexible there can be no doubt.

And there can be no doubt that we will neither survive as individuals nor endure as organizations if we fail to take these most basic and necessary of steps.

Footnotes

[i] Organizations as diverse as the Communist Party, GreenPeace , the Catholic Church and the Tea Party all have such mechanisms as well a written ideology and accompanying rules and regulations.

[ii] The Cryonics Institute (CI) has historically operated on a the “strong leader” paradigm wherein a single individual, or at most a few individuals, determine the proper course for the organization and make decisions about what is just, ethical and moral on case by case or ad hoc basis.

IV. EFFECTS OF CRYOPRESERVATION ON THE HISTOLOGY OF SELECTED TISSUES (Left Ventricle and Cerebral Cortex)

Left Ventricle

Figure 43: The myofibrils of each cardiac muscle cell are branched and contain a single nucleus. The branches interlock with those of adjacent fibers by adherens junctions which act to prevent scission of the cardiomycytes during the high-shear, forceful contractions of the heart. The muscle is richly supplied with mitochondria which are largely confined to the spaces between the fibrils. The fibrils are covered with a membrane, the Sarcolemma, which is frequently invaginated to form the Transverse tubules. These invaginations of the plasma membrane or sarcolemma, are called transverse tubules and they reach deep into the myofibrils and bring the action potential deep into the fibers. Specialized intercellular junctions, the Intercalated discs, facilitate rapid transmission of the electrical signals which initiate myocyte contraction. The myofibrils are formed by myosin and actin fibers aligned in a distinct pattern which is visible under light microscopy as the A-, H- and I- bands.

Yajima stain was used to prepare the Control (Figure 44), FGP and FIG cardiac tissue for light microscopy. The FGP cardiac muscle showed increased interstitial space, probably indicative of interstitial edema. In many areas the sarcolemma appeared to be separated from the cytoplasm of the myocyte and, occasionally, appeared to have disintegrated into debris in the interstitial spaces (Figure 45). The myofilaments appeared maximally relaxed with widened I-bands . The mitochondria were grossly swollen and contained numerous amorphous matrix densities. The sarcolemma was fragmented beneath an intact basement membrane and there was increased space between the capillary endothelium/basement membrane and intact areas of the sarcolemma of the cardiomyocytes. The cell nuclei were unremarkable.

Figure 44:Control-1, Left Ventricle, Yajima, 100x. Control cardiac muscle demonstrated crisp, well defined membranes and the normal density and pattern of myofibril structure. Capillary endothelium appeared intact and the capillary basement membrane was well anchored to adjacent myocytes and appeared intact.

Figure 45:FGP-1 Left Ventricle, Yajima, 100x. In the FGP animals the myocardium exhibited increased interstitial space (IIS) as well as the presence of debris in the IIS which appeared to be disrupted sarcolemma (yellow arrows). The capillary basement membrane was often observed to be separated from the sarcolemma of the adjacent myocytes and endothelial cell nuclei were sometimes observed devoid of plasma membranes or cytoplasm (red arrow).The occasional naked myocyte nucleus could also be observed (green arrow).

The same changes were also present in the FIG group with the added presence of a “ragged” or rough appearance of the myofibrils where they were silhouetted against interstitial space (Figure 46). There also appeared to be holes or spaces, possibly as a result of edema, in the fabric of the myofibrils that were not present in the myocardium of either the control, or the FGP animals.

Most surprising was the general absence of contraction band necrosis in the FIG group, possibly as a consequence of the protective effect of reasonably prompt post-cardiac arrest refrigeration. No microscopic evidence of fracturing, either gross or microscopic, was noted in the myocardium of either the FGP, or the FIG groups.

Figure 46:FIG-2 Left Ventricle, Yajima, 100x. Separation and fragmentation of the sarcolemma were observed in the FIG myocardium to a greater extent than that seen the in myocardium from the FGP animals (yellow arrow). Additionally, the fibers of myofibrils had a more ragged appearance and consistently displayed open spaces in the bands which were not seen in the myocardium of either the Control or the FGP animals (red arrows).

Figure 47: The myofibrils of both the FGP and FIG animals appeared maximally relaxed with a marked increase in the thickness of the I-band. Intact red blood cells (RBCs) were observed in the FIG animals and represent incomplete blood washout (red cell trapping) despite perfusion with large volumes of washout, cryoprotectant and fixative solution (~8-10 L) over a time course of ~140 minutes of perfusion.

Cerebral Cortex

Figure 48: The cerebral cortex consists of six distinct layers, beginning with the first layer, the Molecular Layer (Stratum zonale), which consists of finely branched medullated and non-medullated nerve fibers. The molecular layer is largely devoid of neuronal cell bodies. Those neuronal cell bodies which are present are the cells of Cajal which possess irregular cell bodies and typically have four or five dendrite that terminate within the molecular layer and a long nerve fiber process, or neuraxon, which runs parallel to the surface of the cortical convolutions.

The second layer of the cortex consists of a layer of small Pyramidal cells with the apices of the pyramids being directed towards the surface of the cortex. The apex of the small Pyramidal cells terminates in a dendron, which reaches into the molecular layer, giving off several collateral horizontal branches. The final branches in the molecular layer take a direction parallel to the surface. Smaller dendrites arise from the lateral and basal surfaces of these cells, but do not extend far from the body of the cell. The neuronal axon (neuraxon) always arises from the base of the small Pyramidal cells and passes towards the central white matter, thus forming one of the nerve-fibers of the white matter. In its path, the neuraxon gives off a number of collaterals at right angles, which are distributed to the adjacent grey matter.

The third cortical layer consists of Pyramidal neurons which are characterized by the presence of cells of the same type as those of the preceding layer, but of a larger size. The nerve-fiber process becomes a medullated fiber of the white matter.

The fourth layer is comprised of Polymorphous neurons which are irregular in outline and give off several dendrites which branch into the surrounding grey matter. The neuraxons of the Polymorphous neurons give off a number of collaterals, and then become a nerve-fiber of the central white matter. Scattered through these three layers are the cells of Golgi, whose neuraxon divides immediately with the divisions terminating in the immediate vicinity of the Polymorphous neuron cell-bodies. Some cells are also found in which the neuraxon, instead of extending into the white matter of the brain, passes towards the surface of the cortex; these are called cells of Martinotti.

The fifth cortical layer contains the largest pyramidal neurons which send outputs to the brain stem and spinal cord and comprise the the pyramidal tract. Layer 5 is particularly well-developed in the motor cortex.

Layer 6 consists of pyramidal neurons and neurons with spindle-shaped cell bodies. Most cortical outputs leading to the thalamus originate in layer 6, whereas most outputs to other subcortical nuclei originate in layer 5.

The cortical blood supply is via the pia mater which overlies the cerebral hemispheres.

Bodian stain was used to prepare the control, FGP, and FIG brain tissue samples for light microscopy. Three striking changes were apparent in FGP cerebral cortex histology: 1) marked dehydration of both cells and cell nuclei, 2) the presence of tears or cuts at intervals of 10 to 30 microns throughout the tissue on a variable basis (some areas were spared while others were heavily lesioned), and 3) the increased presence (over Control) of irregular, empty spaces in the neuropil as well as the occasional presence of large peri-capillary spaces (Figures 54,56, and 57). These changes were fairly uniform throughout both the molecular layer and the second layer of the cerebral cortex. Changes in the white matter paralleled those in the cortex, with the notable exception that dehydration appeared to be more pronounced (Figure 55).

Other than the above changes, both gray and white matter histology appeared remarkably intact, and only careful inspection could distinguish it from control (Figures 52, 58, 59 and 60). The neuropil appeared normal (aside from the aforementioned holes and tears) and many long axons and collaterals could be observed traversing the field. Cell membranes appeared crisp, and apart from appearing dehydrated, neuronal architecture appeared comparable to control. Similarly, staining was comparable to that observed in Control cerebral cortex. Cell-to-cell connections appeared largely intact.

The histological appearance of FIG brain differed from that of FGP animals in that ischemic changes such as the presence of pyknotic and fractured nuclei were much in evidence and cavities and tears in the neuropil appeared somewhat more frequently. The white matter of the FIG animals presented a macerated appearance, in addition to exhibiting the rips or tears observed in the white matter of the FGP brains (Figure 61).

Figure 52:FGP-1, Cerebral Cortex, 1st cell layer, Bodian, 40x. Two large capillaries (LC) are present, one with a red blood cell present (right). Neurons (N, cells of Cajal) are present in normal density and the neuropil appears intact. This section appears indistinguishable from that of the Control animal.

Figure 53:FGP-1, Cerebral Cortex, 2nd cell layer, Bodian, 40x. This area of FGP cerebral cortex shows injury typical of that seen in both FGP and FIG animals. There are a number of large tears in the neuropil (red arrows) approximately 10 to 30 microns across. A pyramidal neuron is present in the lower left of the micrograph and it appears somewhat dehydrated. There are a number of naked glial cell nuclei (yellow arrows), as well some nuclei with what appears to adherent cytoplasm visible at the margins of the tears in the neuropil.

Figure 54:FGP-1, Cerebral Cortex, 2nd cell layer, Bodian, 40x. In this area of the 2nd layer of the cerebral cortex the neuropil presents a somewhat “moth eaten” appearance, with numerous tears and vacuoles in evidence (red arrows). One large tear appears to be a pericapillary ice hole (yellow arrow).

Figure 55:FGP-3, Cerebral Cortex, white matter, Bodian, 100x. There are numerous open spaces in the white matter that appear to be ice holes (red arrows). The density of the tissue appears markedly increased over that of the Control white matter, possibly as a result of glycerol-induced dehydration. This apparent dehydration is also evident in the increased density of the axoplasm seen in the myelinated axons (green arrows).

Figure 57:FIG-2, Cerebral Cortex, 1st cell layer, Bodian, 40x. Large tears are evident (red arrows) and naked glial cell nuclei and fragmented cytoplasm are apparent (nn). Several intact capillaries are in evidence (C) as well as what appears to be two capillaries that have been separated from the neuropil and appear largely surrounded by open (pericapillary) space (green arrows). A mass of debris appears to occupy some of the luminal space of what appears to have been a capillary (Cd).

Figure 58:FIG-2, Cerebral Cortex, 2nd cell layer, Bodian, 40x. Remarkably intact neuropil with several capillaries, including several capillaries sectioned oblique to the plane of the tissue (OC). A neuron (N) with what appears to be a crisp plasma membrane is present at the upper right of the micrograph.

Figure 61:FIG-2, Cerebral Cortex, white matter, Bodian, 40x. Severely injured white matter typical of that seen in FIG animals. The tissue presents a macerated appearance (black circles) with numerous rips and tears, possibly as a result of ice formation (red arrows). The capillaries (C) are separated from the tissue parenchyma (yellow arrow) and what appears to be a naked endothelial cell nuclei projected into the intraluminal space of one capillary (green arrow).

I have been traveling, or here at Krell House in Northern Arizona, since 24 December of 2011, and have had virtually no access to the Internet, or to long distance telephony during that time. Additionally, communicating with the world via Chronosphere or email have been comparatively low priorities.

The extreme daily hysteresis in the ambient temperature and humidity in Northern Arizona rapidly degrades coatings and causes the underlying structures to fall to ruin. One example of what I am doing to defend against this is to protect high damage areas of buildings with FRP (fiberglass reinforced paneling) treated with a UV protective coating (photo above). I might also add that these conditions make the use of nails in wood construction inadvisable. Within the space of a year nails, even under painted surfaces, will be extruded ~2-3 mm from the lumber they are embedded in. After ~5 years, they may have backed out of the wood so much as a result of the relentless daily cycles of expansion and contraction (of the wood) that they simply fall out! Screws and glues are the only way to build here.

I arrived in Arizona to find serious damage to the roof of my home, as well as a large number of deferred maintenance tasks crying out for completion. I also discovered that the phone/internet access cable to my home, as well as the fiber optic trunk, had been accidentally attacked by a neighbor’s backhoe. In fact, over 100 ft of phone cable had been uprooted from the ground and requires reburial – a task I’m attending to now.

The telephone cable to my home was uprooted from a point on the adjacent property right up the junction box where it enters the house.

Added food, emergency lighting, and other reserves (above).

An additional 1,000 gallons of water has been brought on line and connected to the house pumping system (above) for a total capacity of 3,000 gallons.

As the world economic and political situation continues its decline I am also increasingly working to prepare for the likelihood of even harder time ahead. I have increased long term food reserves, added an additional 1,000 gallons of water storage capacity and implemented a crude rainwater collection system.

Generator,house power interface (above).

I’ve also completed installation of the back up generator switchover system which allows a seamless (and safe) transition of the house from grid to 5 kw of generator power. I am currently working on the support systems for a small (~ 250 watt) solar panel/battery bank system (battery house, charge controller and inverter)

Heavily insulated and heated battery shed.

Another high priority has been to create the infrastructure required to allow year round cultivation of greens and root vegetables. As a child, I was responsible for maintaining our two “hotbeds” which provided our family with Bibb lettuce, Musclun, bunching onions and salad lettuce for most of the winter. That system relied on fermenting manure in a glass covered wooden frame that was largely buried in the ground to provide both heat and fertilizer.

Unfortunately, the large hysteresis in daily temperature here, coupled with the presence of abundant sunlight, creates real problems for that system of cultivation absent nearly constant attention. Ambient temperature typically fluctuates between 50 to 60 degrees F during the day, to as low as the teens or low 20s at night. Days are often cloudless and bright which means that the temperature in any kind of glassed-in enclosure could easily and rapidly exceed 120 degrees F! Thus, such an enclosure would have to be opened and closed at least twice a day; with any failure to do so likely resulting in the loss of the crops.

Initial excavation and stone-laying of the cultivation chamber. The tank visible in the background is a 1,000 gallon propane tank.

Nearly completed stonework with finished grading.

Until very recently, scrap Kaibab stone was available free here. Even now, it is only $20.00 for a level pick-up truck bed full. This has allowed me to construct a large, well insulated, earth sheltered and heat-sink protected cultivation chamber.

Construction is well underway on the sunlight admitting glass and environmental module that will be bolted to the stonework. The cross members seen in the photo above will soon be decorated with an automatic, solar powered climate control system. When the internal temperature exceeds the safe limit, a muffin fan is activated to bring in cooler air from the outside. In the summer, cool, moist air is generated and delivered via an underground network of pipes that also uses evaporative cooling. Watering is also automatically controlled. The entire set up was built to be resistant to penetration by radionuclides. This is a far more difficult challenge than keeping a stock of soil protected (which can be done quite simply by using earth covered tarps).

This project has been an especially high priority for me because it is no longer economically possible for me to have access to fresh greens, or similar, highly perishable vegetables. I live an hour’s drive from any affordable shopping of this kind, and the prices of these items has also skyrocketed. With consumer petrol prices predicted to be near $5.00 per gallon by this summer, I will have to reduce resupply trips from every two weeks to once a month – and possibly longer. I believe that the prolonged absence of this kind of food from the diet is a serious health risk – as well as being an added misery.

Another food related project is the construction of a chicken enclosure…

Heat wasting windows have been “replaced” with high grade insulation and firewood stores have been increased (above). The porch light is a high output, high efficiency LED bulb (60K hour life) brought back from Europe along with a suitcase full of others! The yellow coating was done by me.

Wood, like stone, is abundant here, and for a small fee to the Forrest Service it is possible to cut a great deal for the cost of the time, gasoline and wear and tear on the chain saw. I have thus increased my firewood reserves, and plan to increase them further. Sometime ago I “eliminated” all the windows in my home, or more properly, heavily insulated them with expanded polystyrene and fiberglass faced with a double sided, multilayer reflective heat barrier. This has reduced heating and cooling costs by over 90%. I am staggered at the massive amount of heat leak that occurs through so called “energy efficient” double pane windows. Most people aren’t in their homes during the daytime and when they are they are usually watching TV or on the computer. LED lighting is now so cheap (in Europe) that it is vastly more economical to light your home with electricity and dispense with energy gobbling windows altogether. If you need to look outside – well, that’s what cameras are for – and they can see in the infrared too, which means you can see what’s going on in the dark.

Chronospohere, at least as it has been pursued so far, has failed to gain traction. I will explore what I think are the reasons for this at a later date.

For the present, I am busy and productive and working within my (small) resource constraints. Progress is slow because almost everything I do is on a no cash basis using items recovered from the waste stream, bartered for, or purchased as scrap for one cent on the dollar (or less). It is also slow, since I am doing it myself and learning as I go along. I am blessed with a good library of books on everything from electrical wiring to woodworking. The only books thrown out more consistently in the UK and the US than the Bible are ‘self help’ and ‘how to’ books. I am becoming increasingly convinced that many people buy such books with the expectation that merely owning them will somehow magically confer mastery of their contents. Probably the same is also true of the Bible.

I am attending to the large backlog of personal correspondence that has accumulated during my period of enforced isolation from the Internet, so, if you have written me, I apologize for the tardiness of my response in advance. — Mike Darwin

IV. EFFECTS OF CRYOPRESERVATION ON THE HISTOLOGY OF SELECTED TISSUES (Liver and Kidneys)

Histology was evaluated in two animals each from the FIG and FIGP groups, and in one control animal. Only brain histology was evaluated in the straight-frozen control animal.

Liver

The histological appearance of the liver in all three groups of animals was one of profound injury. Even in the FGP group, the cellular integrity of the liver appeared grossly disrupted. In liver tissue prepared using Yajima stain, the sinusoids and spaces of Disse were filled with flocculent debris, and it was often difficult or impossible to discern cell membranes (Figures 30-32). The collagenous supporting structures of the bile canaliculi were in evidence and the nuclei of the hepatocytes appeared to have survived with few alterations evident at the light level, although frequent pyknotic nuclei were noted in the FIGP group (Figures 31 & 34). Indeed, the nuclei often appeared to be floating in a sea of amorphous material (Figure34). Not surprisingly, the density of staining of the cytoplasmic material was noticeably reduced over that of the fixative-perfused control. Few intact capillaries were noted.

FGP liver tissue prepared with PAS stain exhibited a similar degree of disruption (Figure 32). However, quite remarkably, the borders of the hepatocytes were defined by a clear margin between glycogen granule containing cytoplasm and non-glycogen containing membrane or other material (membrane debris?) which failed to stain with Yajima due to gross physical disruption, or altered tissue chemistry (Figure 35).

Figure 27: The fundamental histological structural unit of the liver is the liver lobule, a six-sided prism of tissue ~ 2 mm long and ~1 mm in diameter. The lobule is defined by interlobular connective tissue which is not very visible under light microscopy in the cat (or in man). In the corners of the lobular prisms are the portal triads. In tissue cross sections prepared for microscopy, the lobule is filled by cords of hepatic parenchymal cells, the hepatocytes, which radiate from the central vein and are separated by vascular sinusoids. The bulk of the liver consists of epithelial hepatocytes arranged into cords, separated by the vascular sinusoids through which the portal blood percolates. The epithelium of the sinusoids is decorated with phagocytic Kuppfer cells that are the primary mechanism for removing gut bacteria present in the venous splanchnic circulation.

The cords of hepatocytes comprise the hepatic parenchyma. In section, the hepatic cords appear as linear ropes (or cords) of hepatocytes. Viewed 3-dimensionaly, the cords consist of intricately folded branching and connected planes of cells which extend parallel to the long axis of the lobule and radiate out from the its center. The hepatocytes in each cord are attached to each other wherever they come into contact, as well as to the sinusoids at either end of the lobular pyramid. The sinusoids are vascular spaces lined by fenestrated endothelium that has no basement membrane, thus allowing the plasma to pass over the large surface area sheets of hepatocytes for detoxification. The sinusoid endothelium stands off from the underlying hepatocytes allowing space for the plasma to interact with the hepatocytes and Kupffer cells (the space of Disse).

Bile canaliculi, formed by apical surfaces of adjacent hepatocytes, form a network of tiny passages contained within each hepatic cord.

Figure 28: Control-1 Liver, Yajima, 100x. Liver sections from the Control animal demonstrated normal morphology as can be seen in the image above.

Figure 29:Control-1 Liver, PAS, 100x. Liver sections were prepared with both Yajima and PAS stain in order to allow visualization of structures that neither stain discloses alone; in this case, most importantly, the presence or glycogen granules in the hepatocytes of the Control animal. Note the presence of normal intralobular architecture with crisp cell membranes in evidence, normal appearing sinusoid spaces, and residual sinusoidal red blood cells (RBCs) not washed out during fixative perfusion.

Figure 30:FGP-1 Liver, Yajima, 100x. The livers of FGP animals demonstrated extensive histological disruption. The sinusoids were all but obliterated and appeared filled with debris (ds) and the cytoplasm was extensively vacuolated (v).

Figure 31:FIG-2 Liver, Yajima, 100x. As was the case with the FGP animals, the sinusoids were barely discernable and appeared filled with cellular debris (cd). In addition to extensive cytoplasmic (cv) and nuclear vacuolization (nv), pyknotic nuclei (pn) were also present. Cell membranes were difficult to discern and in many areas, frank cell lysis appears to have occurred with flocculent cellular debris (cd) appearing to fill the sinusoids.

Figure 32:FGP-1, Liver, PAS, 100x. The intensely red-stained granules present in the cytoplasm of the hepatocytes are glycogen deposits selectively stained by PAS. There is extensive cytoplasmic (cv) and nuclear vacuolization (nv) and the sinusoids appear filled with flocculent cellular debris (d). Indeed, it is only possible to discern the outlines of the original individual hepatocytes from the pattern of the intracellular glycogen granules disclosed by the PAS stain.

Figure 33: FIG-2, Liver, Yajima, 100x, well preserved area. While the bulk of the hepatic parenchyma exhibited the severe injury seen in Figures 30-32, there were frequently observed islands of comparatively well preserved tissue visible in both the FGP and FIGP sections suggesting that freezing injury is occurring non-homogenously.

Figure 34: FIG-2, Liver, PAS, 100x, necrotic area. There were patchy areas of frank necrosis visible in the livers of the FIGP animals that were not present in the livers of the FGP animals. This area, adjacent to a central vein, shows extensive cell lysis with heavy vacuolization of the cytoplasm (v) and many pyknotic nuclei (pn) in evidence.

Figure 35: FIG-2, Liver, PAS, 100x. Note the presence of a few scattered glycogen granules (GG). Interestingly, in this comparatively well preserved area of FIGP liver it is possible to see some remaining deposits of glycogen that were not consumed during the long post-arrest ischemic interval. The absence of pyknotic nuclei and the relative absence of large intracellular vacuoles is also remarkable.

Kidney

Figure 36: The functional unit of the kidney is the nephron, consisting of the glomerulus and the uriniferous tubule ( the renal corpuscle: a).The capillary tuft of the nephron, the glomerulus, is enclosed within a double cell layered structure; Bowman’s capsule. Bowman’s capsule and the capillary tuft it encloses comprise the glomerulus. Bowman’s capsule and the glomerular capillary tuft constitute the renal (or Malpighian) corpuscle (b).

Bowman’s capsule opens into the proximal convoluted tubule which leads to the loop of Henle. The loop of Henle leads to the distal convoluted tubule which then leads to the collecting duct.

The inner layer of Bowman’s capsule is the visceral layer. It consists of cells called podocytes. The outer layer of Bowman’s capsule is the parietal layer. The pedicels are the foot processes on the podocytes.

The juxtaglomerular cells secrete renin which is ultimately metabolized into angiotensin II, a potent vasoconstrictor critical to maintaining normotension. The macula densa are specialized cells in the distal convoluted tubule that are responsible for sodium, and thus fluid regulation. The juxtaglomerular cells and macula densa make up the juxtaglomerular apparatus.

PAS stain was used to prepare the control, FGP and FIGP renal tissue for light microscopy. The histological appearance of FGP renal tissue was surprisingly good (Figures 329, 40 & 41). The glomeruli and tubules appeared grossly intact and stain uptake was normal. However, a number of alterations from the appearance of the control were apparent. The capillary tuft of the glomeruli appeared swollen and the normal space between the capillary tuft and Bowman’s capsule was absent. There was also marked interstitial edema, and marked cellular edema as evidenced by the obliteration of the tubule lumen by cellular edema.

By contrast, the renal cortex of the FIGP animals, when compared to either the control or the FGP group, showed a profound loss of detail, absent intercellular space, and altered staining (Figures 40 & 42). The tissue appeared frankly necrotic, with numerous pyknotic nuclei and numerous large vacuoles which peppered the cells. One striking difference between FGP and FIGP renal cortex was that the capillaries, which were largely obliterated in the FGP animals, were consistently spared in the FIGP animals. Indeed, the only extracellular space in evidence in this preparation was the narrowed lumen of the capillaries, grossly reduced in size apparently as a consequence of cellular edema.

Figure 39:FGP-1, Renal Cortex, PAS, 40x. The intertubular space (ITS) is great expanded and the tubule cells are heavily vacoulated (V) and lack definition. The intratubular space (IS) is no longer in evidence and the architecture of the glomerluar capillary tuft (GT) is radically altered and there is an absence of the normal architecture of Bowman’s space (yellow arrows). The intertubular capillaries appear to have been reduced to debris (D) visible in the intertubular spaces.

Figure 41:FIG-2 Renal Cortex, fracture present (arrows), PAS, 40x. Two renal tubules, possibly a proximal and distal convoluted tubule (T) are dissected by a fracture as is the macula densa (MD) of the glomerulus (G). Remarkably, there is still a small amount of intertubular space present in this micrograph.

The immediate goal of human cryopreservation is to use current cryobiological techniques to preserve the brain structures which encode personal identity adequately enough to allow for resuscitation or reconstruction of the individual should molecular nanotechnology be realized (1,2). Aside from two previous isolated efforts (3,4) there has been virtually no systematic effort to examine the fidelity of histological, ultrastructural, or even gross structural preservation of the brain following cryopreservation in either an animal or human model. While there is a substantial amount of indirect and fragmentary evidence in the cryobiological literature documenting varying degrees of structural preservation in a wide range of mammalian tissues (5,6,7), there is little data of direct relevance to cryonics. In particular, the focus of contemporary cryobiology has been on developing cryopreservation techniques for currently transplantable organs, and this has necessarily excluded extensive cryobiological investigation of the brain, the organ of critical importance to human identity and mentation.

The principal objective of this pilot study was to survey the effects of glycerolization, freezing to liquid nitrogen temperature, and rewarming on the physiology, gross structure, histology, and ultrastructure of both the ischemic and non-ischemic adult cats using a preparation protocol similar to the one then in use on human cryopreservation patients. The non-ischemic group was given the designation Feline Glycerol Perfusion (FGP) and the ischemic group was referred to as Feline Ischemic Glycerol Perfusion (FIGP).

The work described in this paper was carried out over a 19-month period from January, 1982 through July, 1983. The perfusate employed in this study was one which was being used in human cryopreservation operations at that time, the composition of which is given in Table I.

The principal cryoprotectant was glycerol.

II. MATERIALS AND METHODS

Pre-perfusion Procedures

Nine adult cats weighing between 3.4 and 6.0 kg were used in this study. The animals were divided evenly into a non-ischemic and a 24-hour mixed warm/cold ischemic group. All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the National Institutes of Health (NIH Publication No. 80-23, revised 1978). Anesthesia in both groups was secured by the intraperitoneal administration of 40 mg/kg of sodium pentobarbital. The animals were then intubated and placed on a pressure-cycled ventilator. The EKG was monitored throughout the procedure until cardiac arrest occurred. Rectal and esophageal temperatures were continuously monitored during perfusion using YSI type 401 thermistor probes.

Following placement of temperature probes, an IV was established in the medial foreleg vein and a drip of Lactated Ringer’s was begun to maintain the patency of the IV and support circulating volume during surgery. Premedication (prior to perfusion) consisted of the IV administration of 1 mg/kg of metubine iodide to inhibit shivering during external and extracorporeal cooling and 420 IU/kg sodium heparin as an anticoagulant. Two 0.77 mm I.D. Argyle Medicut 15″ Sentinel line catheters with Pharmaseal K-69 stopcocks attached to the luer fittings of the catheters were placed in the right femoral artery and vein. The catheters were connected to Gould Model P23Db pressure transducers and arterial and venous pressures were monitored throughout the course of perfusion.

Surgical Protocol

Following placement of the monitoring catheters, the animals were transferred to a tub of crushed ice and positioned for surgery. The chest was shaved and a median sternotomy was performed. The aortic root was cleared of fat and a purse-string suture was placed, through which a 14-gauge Angiocath was introduced. The Angiocath, which served as the arterial perfusion cannula, was snared in place, connected to the extracorporeal circuit and cleared of air. The pericardium was opened and tented to expose the right atrium. A purse-string suture was placed in the apex of the right atrium and a USCI type 1967 16 fr. venous cannula was introduced and snared in place. Back-ties were used on both the arterial and venous cannulae to secure them and prevent accidental dislodgment during the course of perfusion. Placement of cannulae is shown in Figure 1.

Figure 1: Vascular access for extracorporeal perfusion was via median sternotomy. The arterial cannula consisted of a14-gauge Angiocath (AC) which was placed in the aortic root (AR) and secured in place with a purse string suture. A USCI type 1967 16 fr. venous cannula (VC) was placed in the right atrium (RA) and snared in place using 0-silk ligature and a length of Red Robinson urinary catheter (snare). The chest wound was kept open using a Weitlander retractor. The left ventricle (LV) was not vented.

The extracorporeal circuit (Figures 2&3) was of composed of 1/4″ and 3/8″ medical grade polyvinyl chloride tubing. The circuit consisted of two sections: a recirculating loop to which the animal was connected and a glycerol addition system. The recirculating system consisted of a 10 liter polyethylene reservoir positioned atop a magnetic stirrer, an arterial (recirculating) roller pump, an Erika HPF-200 hemodialyzer which was used as a hollow fiber oxygenator (8) (or alternatively, a Sci-Med Kolobow membrane oxygenator), a Travenol Miniprime pediatric heat exchanger, and a 40-micron Pall LP 1440 pediatric blood filter. The recirculating reservoir was continuously stirred with a 2″ Teflon-coated magnetic stir bar driven by a Corning PC 353 magnetic stirrer. Temperature was continuously monitored in the arterial line approximately 15.2 cm from the arterial cannula using a Sarns in-line thermistor temperature probe and YSI 42SL remote sensing thermometer. Glycerol concentrate was continuously added to the recirculating system using a Drake-Willock dual raceway hemodialysis pump, while venous perfusate was concurrently withdrawn from the circuit and discarded using a second raceway in the same pump head.

Figure 3: Schematic of cryoprotective perfusion circuit.

Storage and Reuse of the Extracorporeal Circuit

After use the circuit was flushed extensively with filtered tap and distilled water, and then flushed and filled with 3% formaldehyde in distilled water to prevent bacterial overgrowth. Prior to use the circuit was again thoroughly flushed with filtered tap water, and then with filtered distilled water (including both blood and gas sides of the hollow fiber dialyzer; Kolobow oxygenators were not re-used). At the end of the distilled water flush, a test for the presence of residual formaldehyde was performed using Schiff’s Reagent. Prior to loading of the perfusate, the circuit was rinsed with 10 liters of clinical grade normal saline to remove any particulates and prevent osmotic dilution of the base perfusate.

Pall filters and arterial cannula were not re-used. The circuit was replaced after a maximum of three uses.

Preparation of Control Animals

Fixative Perfusion

Two control animals were prepared as per the above. However, the animals were subjected to fixation after induction of anesthesia and placement of cannulae. Fixation was achieved by first perfusing the animals with 500 mL of bicarbonate-buffered Lactated Ringer’s containing 50 g/l hydroxyethyl starch (HES) with an average molecular weight of 400,000 to 500,000 supplied by McGaw Pharmaceuticals of Irvine, Ca (pH adjusted to 7.4) to displace blood and facilitate good distribution of fixative, followed immediately by perfusion of 1 liter of modified Karnovsky’s fixative (Composition given in Table I). Buffered Ringers-HES perfusate and Karnovsky’s solution were filtered through 0.2 micron filters and delivered with the same extracorporeal circuit described above.

Immediately following fixative perfusion the animals were dissected and 4-5 mm thick coronal sections of organs were cut, placed in glass screw-cap bottles, and transported, as detailed below, for light or electron microscopy.

Straight Frozen Non-ischemic Control

One animal was subjected to straight freezing (i.e., not treated with cryoprotectant). Following induction of anesthesia and intubation the animal was supported on a ventilator while being externally cooled in a crushed ice-water bath. When the EKG documented profound bradycardia at 26°C, the animal was disconnected from the ventilator, placed in a plastic bag, submerged in an isopropanol cooling bath at -10°C, and chilled to dry ice and liquid nitrogen temperature per the same protocol used for the other two experimental groups as described below.

Preparation of FGP Animals

Following placement of cannulae, FGP animals were subjected to total body washout (TBW) by open-circuit perfusion of 500 mL of glycerol-free perfusate. The extracorporeal circuit was then closed and constant-rate addition of glycerol-containing perfusate was begun.

Cryoprotective perfusion continued until the target concentration of glycerol was reached or the supply of glycerol-concentrate perfusate was exhausted.

Preparation of FIGP Animals

In the FIGP animals, ventilator support was discontinued following anesthesia and administration of Metubine. The endotracheal tube was clamped and the ischemic episode was considered to have begun when cardiac arrest was documented by absent EKG.

After the start of the ischemic episode the animals were allowed to remain on the operating table at room temperature ( 22°C to 25°C) for a 30 minute period to simulate the typical interval between pronouncement of legal death in a clinical environment and the start of external cooling at that time. During the 30 minute normothermic ischemic interval the femoral cut-down was performed and monitoring lines were placed in the right femoral artery and vein as per the FGP animals. Prior to placement, the monitoring catheters were irrigated with normal saline, and following placement the catheters were filled with 1000 unit/mL of sodium heparin to guard against clot obstruction of the catheter during the post-arrest ischemic period.

After the 30 minute normothermic ischemic period the animals were placed in a 1-mil polyethylene bag, transferred to an insulated container in which a bed of crushed ice had been laid down, and covered over with ice. A typical cooling curve for a FIGP animal is presented in Figure 4. FIGP animals were stored on ice in this fashion for a period of 24 hours, after which time they were removed from the container and prepared for perfusion using the surgical and perfusion protocol described above.

Perfusate

TABLE I

Perfusate Composition

Component mM

Potassium Chloride 2.8

Dibasic Potassium Phosphate 5.9

Sodium Bicarbonate 10.0

Sodium Glycerophosphate 27.0

Magnesium Chloride 4.3

Dextrose 11.0

Mannitol 118.0

Hydroxyethyl Starch 50 g/l

The perfusate was an intracellular formulation which employed sodium glycerophosphate as the impermeant species and hydroxyethyl starch (HES)(av. MW 400,000 - 500,000) as the colloid. The composition of the base perfusate is given in Table I. The pH of the perfusate was adjusted to 7.6 with potassium hydroxide. A pH above 7.7, which would have been “appropriate” to the degree of hypothermia experienced during cryoprotective perfusion (9), was not achievable with this mixture owing to problems with complexing of magnesium and calcium with the phosphate buffer, resulting in an insoluble precipitate.

Perfusate components were reagent or USP grade and were dissolved in USP grade water for injection. Perfusate was pre-filtered through a Whatman GFB glass filter (a necessary step to remove precipitate) and then passed through a Pall 0.2 micron filter prior to loading into the extracorporeal circuit.

Perfusion

Perfusion of both groups of animals was begun by carrying out a total body washout (TBW) with the base perfusate in the absence of any cryoprotective agent. In the FGP group washout was achieved within 2 – 3 minutes of the start of open circuit asanguineous perfusion at a flow rate of 160 to 200 mL/min and an average perfusion pressure of 40 mm Hg. TBW in the FGP group was considered complete when the hematocrit was unreadable and the venous effluent was clear. This typically was achieved after perfusion of 500 mL of perfusate.

Complete blood washout in the FIGP group was virtually impossible to achieve (see “Results” below). A decision was made prior to the start of this study (based on previous clinical experience with ischemic human cryopreservation patients) not to allow the arterial pressure to exceed 60 mm Hg for any significant period of time. Consequently, peak flow rates obtained during both total body washout and subsequent glycerol perfusion in the FIGP group were in the range of 50-60 mL/min at a mean arterial pressure of 50 mm Hg.

Due to the presence of massive intravascular clotting in the FIGP animals it was necessary to delay placement of the atrial (venous) cannula (lest the drainage holes become plugged with clots) until the large clots present in the right heart and the superior and inferior vena cava had been expressed through the atriotomy. The chest was kept relatively clear of fluid/clots by active suction during this interval. Removal of large clots and reasonable clearing of the effluent was usually achieved in the FIGP group after 15 minutes of open circuit asanguineous perfusion, following which the circuit was closed and the introduction of glycerol was begun.

Figure 5: pH of non-ischemic Δ•▪*(FGP) and ischemic ●●●ᴑ (FIGP) cats during cryoprotective perfusion. The FIGP animals were, as expected, profoundly acidotic with the initial arterial pH being between 6.5 and 6.6.

The arterial pO2 of animals in both the FGP and FIGP groups was kept between 600 mm Hg and 760 mm Hg throughout TBW and subsequent glycerol perfusion. Arterial pH in the FGP animals was between 7.1 and 7.7 and was largely a function of the degree of diligence with which addition of buffer was pursued. Arterial pH in the FIGP group was 6.5 to 7.3. Two of the FIGP animals were not subjected to active buffering during perfusion and as a consequence recovery of pH to more normal values from the acidosis of ischemia (starting pH for FIGP animals was typically 6.5 to 6.6) was not as pronounced (Figure 5).

Introduction of glycerol was by constant rate addition of base perfusate formulation made up with 6M glycerol to a recirculating reservoir containing 3 liters of glycerol-free base perfusate. The target terminal tissue glycerol concentration was 3M and the target time course for introduction was 2 hours. The volume of 6M glycerol concentrate required to reach a terminal concentration in the recirculating system (and thus presumably in the animal) was calculated as follows:

Vp

Mc = ——— Mp

Vc + Vp

where

Mc = Molarity of glycerol in animal and circuit.

Mp = Molarity of glycerol concentrate.

Vc = Volume of circuit and exchangeable volume of animal.*

Vp = Volume of perfusate added.

* Assumes an exchangeable water volume of 60% of the pre-perfusion weight of the animal.

Glycerolization of the FGP animals was carried out at 10°C to 12°C. Initial perfusion of FIGP animals was at 4°C to 5°C with warming (facilitated by TBW with warmer perfusate and removal of surface ice packs) to 10°-12°C for cryoprotectant introduction. The lower TBW temperature of the FIGP animals was a consequence of the animals having been refrigerated on ice for the 24 hours preceding perfusion.

Following termination of the cryoprotective ramp, the animals were removed from bypass, the aortic cannula was left in place to facilitate prompt reperfusion upon rewarming, and the venous cannula was removed and the right atrium closed. The chest wound was loosely closed using surgical staples.

Concurrent with closure of the chest wound, a burr hole craniotomy 3 to 5 mm in diameter was made in the right parietal bone of all animals using a high speed Dremel “hobby” drill. The purpose of the burr hole was to allow for post-perfusion evaluation of cerebralvolume, assess the degree of blood washout in the ischemic animals and facilitate rapid expansion of the burr hole on re-warming to allow for the visual evaluation of post-thaw reperfusion (using dye).

The rectal thermistor probe used to monitor core temperature during perfusion was replaced by a copper/constantan thermocouple at the conclusion of perfusion for monitoring of the core temperature during cooling to -79°C and -196°C.

Cooling to -79°C

Figure 7: Representative cooling curve (esophageal and rectal temperatures) of FGP and FIGP animals from ~ 10°C to ~ -79°C. The ragged curve with sharp temperature excursions and rebounds is an artifact of the manual control of temperature descent via the addition of chunks of dry ice.

Cooling to -79°C was carried out by placing the animals within two 1 mil polyethylene bags and submerging them in an isopropanol bath which had been pre-cooled to -10°C. Bath temperature was slowly reduced to -79°C by the periodic addition of dry ice. A typical cooling curve obtained in this fashion is shown in Figure 7. Cooling was at a rate of approximately 4°C per hour.

Cooling to and Storage at -196°C

Figure 8: Animals were cooled to -196°C by immersion in liquid nitrogen (LN2) vapor in a Linde LR-40 cryogenic dewar. When a core temperature of ~-180 to -185°C was reached, the animals were immersed in LN2.

Following cooling to -79°C, the plastic bags used to protect the animals from alcohol were removed, the animals were placed inside nylon bags with draw-string closures and were then positioned atop a 6″ high aluminum platform in an MVE TA-60 cryogenic dewar to which 2″-3″ of liquid nitrogen had been added. Over a period of approximately 15 hours the liquid nitrogen level was gradually raised until the animal was submerged. A typical cooling curve to liquid nitrogen temperature for animals in this study is shown in Figure 8. Cooling rates to liquid nitrogen temperature were approximately 0.178°C per hour. After cool-down animals were maintained in liquid nitrogen for a period of 6-8 months until being removed and re-warmed for gross structural, histological, and ultrastructural evaluation.

Re-warming

Figure 9: Rewarming of all animals was accomplished by removing the animals from LN2 and placing them in a pre-cooled box insulated with 15.2 cm of polyurethane (isocyothianate) foam to which 1.5 L of LN2 (~2 cm on the bottom of the box) of LN2 had been added. When the core temperature of the animals reached -20°C the animals were transferred to a mechanical refrigerator at 3.4°C.

The animals in both groups were re-warmed to -2°C to -3°C by removing them from liquid nitrogen and placing them in a pre-cooled box insulated on all sides with a 10.2 cm thickness of Styrofoam and containing a small quantity of liquid nitrogen. The animals were then allowed to re-warm to approximately -20°C, at which time they were transferred to a mechanical refrigerator at a temperature of 8°C. When the core temperature of the animals had reached -2°C to -3°C the animals were removed to a bed of crushed ice for dissection, examination and tissue collection for light and electron microscopy. A typical re-warming curve is presented in Figure 9.

Modification of Protocol Due To Tissue Fracturing

After the completion of the first phase of this study (perfusion and cooling to liquid nitrogen temperature) the authors had the opportunity to evaluate the gross and histological condition of the remains of three human cryopreservation patients who were removed from cryogenic storage and converted to neuropreservation (thus allowing for post-arrest dissection of the body, excluding the head) (10). The results of this study confirmed previous, preliminary, data indicative of gross fracturing of organs and tissues in animals cooled to and re-warmed from -196°C. These findings led us to abandon our plans to reperfuse the animals in this study with oxygenated, substrate-containing perfusate (to have been followed by fixative perfusion for histological and ultrastructural evaluation) which was to be have been undertaken in an attempt to assess post-thaw viability by evaluation of post-thaw oxygen consumption, glucose uptake, and tissue-specific enzyme release.

Re-warming and examination of the first animal in the study confirmed the presence of gross fractures in all organ systems. The scope and severity of these fractures resulted in disruption of the circulatory system, thus precluding any attempt at reperfusion as was originally planned.

Preparation of Tissue Samples For Microscopy

Fixation

TABLE II.

Composition Of Modified Karnovsky’s Solution

Component g/l

Paraformaldehyde 40

Glutaraldehyde 20

Sodium Chloride 0.2

Sodium Phosphate 1.42

Calcium Chloride 2.0 mM

pH adjusted to 7.4 with sodium hydroxide.

Samples of four organs were collected for subsequent histological and ultrastructural examination: brain, heart, liver and kidney. Dissection to obtain the tissue samples was begun as soon as the animals were transferred to crushed ice. The brain was the first organ removed for sampling. The burr hole created at the start of perfusion was rapidly extended to a full craniotomy using rongeurs (Figure 14). The brain was then removed en bloc to a shallow pan containing iced, modified Karnovsky’s fixative containing 25% w/v glycerol (see Table II for composition) sufficient to cover it. Slicing of the brain into 5 mm thick sections was carried out with the brain submerged in fixative in this manner. At the conclusion of slicing a 1 mm section of tissue was excised from the visual cortex and fixed in a separate container for electron microscopy. During final sample preparation for electron microscopy care was taken to avoid the cut edges of the tissue block in preparing the Epon embedded sections.

Figure 10: The sagitally sectioned (5 mm thickness) brains of the animals were placed in a perforated basket immersed in Karnofsky’s fixative. This assembly was placed atop a magnetic stirring table and the fixative was gently stirred with a magnetic stirring bar.

The sliced brain was then placed in 350 ml of Karnovsky’s containing 25%w/v glycerol in a special stirring apparatus which is illustrated in Figure 10. This fixation/de-glycerolization apparatus consisted of two plastic containers nested inside of each other atop a magnetic stirrer. The inner container was perforated with numerous 3 mm holes and acted to protect the brain slices from the stir bar which continuously circulated the fixative over the slices. The stirring reduced the likelihood of delayed or poor fixation due to overlap of slices or stable zones of tissue water stratification. (The latter was a very real possibility owing to the high viscosity of the 25%w/v glycerol-containing Karnovsky’s.)

De-glycerolization of Samples

Figure 11: Following fixation, the tissues slices of all organs evaluated by microscopy were serially de-glycerolized using the scheme shown above. When all of the glycerol was unloaded from the tissues they were shipped in modified Karnovsky’s to outside laboratories for histological and electron microscopic imaging.

To avoid osmotic shock all tissue samples were initially immersed in Karnovsky’s containing 25%w/v glycerol at room temperature and were subsequently de-glycerolized prior to staining and embedding by stepwise incubation in Karnovsky’s containing decreasing concentrations of glycerol (see Figure 11 for the de-glycerolization protocol).

To prepare tissue sections from heart, liver, and kidney for microscopy, the organs were first removed en bloc to a beaker containing an amount of ice-cold fixative containing 25% w/v glycerol sufficient to cover the organ. The organ was then removed to a room temperature work surface at where 0.5 mm sections were made with a Stadie-Riggs microtome. The microtome and blade were pre-wetted with fixative, and cut sections were irrigated from the microtome chamber into a beaker containing 200 ml of room-temperature fixative using a plastic squeeze-type laboratory rinse bottle containing fixative solution. Sections were deglycerolized using the same procedure previously detailed for the other slices.

Osmication and Further Processing

At the conclusion of de-glycerolization of the specimens all tissues were separated into two groups; tissues to be evaluated by light microscopy, and those to be examined with transmission electron microscopy. Tissues for light microscopy were shipped in glycerol-free modified Karnovsky’s solution to American Histolabs, Inc. in Rockville, MD for paraffin embedding, sectioning, mounting, and staining.

Tissues for electron microscopy were transported to the facilities of the University of California at San Diego in glycerol-free Karnovsky’s at 1° to 2°C for osmication, Epon embedding, and EM preparation of micrographs by Dr. Paul Farnsworth.

Due to concerns about the osmication and preparation of the material processed for electron microscopy by Farnsworth, tissues from the same animals were also submitted for electron microscopy to Electronucleonics of Silver Spring, Maryland.

III. EFFECTS OF GLYCEROLIZATION

Perfusion of FGP Animals

Blood washout was rapid and complete in the FGP animals and vascular resistance decreased markedly following blood washout. Vascular resistance increased steadily as the glycerol concentration increased, probably as a result of the increasing viscosity of the perfusate.

Within approximately 5 minutes of the beginning of the cryoprotective ramp, bilateral ocular flaccidity was noted in the FGP animals. As the perfusion proceeded, ocular flaccidity progressed until the eyes had lost approximately 30% to 50% of their volume.

Gross examination of the eyes revealed that initial water loss was primarily from the aqueous humor, with more significant losses from the posterior chamber of the eyes apparently not occurring until later in the course of perfusion. Within 15 minutes of the start of glycerolization the corneal surface became dimpled and irregular and the eyes had developed a “caved-in” appearance.

Dehydration was also apparent in the skin and skeletal muscles and was evidenced by a marked decrease in limb girth, profound muscular rigidity, cutaneous wrinkling (Figure 11), and a “waxy-leathery” appearance and texture to both cut skin and skeletal muscle.

Figure 13: Cutaneous dehydration following glycerol perfusion is evidenced by washboard wrinkling of the thoraco-abdominal skin (CD). The ruffled appearance of the fur on the right foreleg (RF) is also an artifact of cutaneous dehydration. The sternotomy wound, venous cannula and the Weitlaner retractor (R) and the retractor blade (RB) holding open the chest wound are visible at the upper left of the photo.

Preliminary observation suggest that water loss was in the range of 30% to 40% in most tissues. As can be seen in Table III, total body water losses attributable to dehydration, while typically not as profound, were still in the range of 18% to 34%. The gross appearance of the heart suggested a similar degree of dehydration, as evidenced by modest shrinkage and the development of a “pebbly” surface texture and a somewhat translucent or “waxy” appearance.

TABLE III.

Total Water-Loss Associated With Glycerolization of the Cat

____________________________________________________

Animal Pre-Perfusion Post-Perfusion Kg./ % Lost As

# Weight Kg. Weight Water Dehydration

FGP-1 4.1 3.6 2.46 18

FGP-2 3.9 3.1 2.34 34

FGP-3 4.5 3.9 2.70 22

FGP-4 6.0 5.0 3.60 28

FIGP-1 3.4 3.0 2.04 18

FIGP-2 3.4 3.2 2.04 9

FIGP-3 4.32 3.57 2.59 29

Figure 14: Cerebrocortical dehydration as a result of 4M glycerol perfusion. The cortical surface (CS) is retracted ~5-8 mm below the margin of the cranial bone (CB).

Examination of the cerebral hemispheres through the burr hole (Figure 14) and of the brain in the brain brainpan (Figure 19) revealed an estimated 30% to 50% reduction in cerebral volume, presumably as a result of osmotic dehydration secondary to glycerolization. The cortices also had the “waxy” amber appearance previously observed as characteristic of glycerolized brains.

The gross appearance of the kidneys, spleen, mesenteric and subcutaneous fat, pancreas, and reproductive organs (where present) were unremarkable. The ileum and mesentery appeared somewhat dehydrated, but did not exhibit the waxy appearance that was characteristic of muscle, skin, and brain.

Figure 15: Oxygen consumption was not apparently affected by glycerolization as can be seen in the data above from the perfusions of FGP-5 and FGP-5.

Oxygen consumption (determined by measuring the arterial/venous difference) throughout perfusion was fairly constant and did not appear to be significantly impacted by glycerolization, as can be seen Figure 12.

Perfusion of FIGP Animals

As previously noted, the ischemic animals had far lower flow rates at the same perfusion pressure as FGP animals and demonstrated incomplete blood washout. Intravascular clotting was serious a barrier to adequate perfusion. Post-thaw dissection demonstrated multiple infarcted areas in virtually all organ systems; areas where blood washout and glycerolization were incomplete or absent. In contrast to the even color and texture changes observed in the FGP animals, the skin of the FIGP animals developed multiple, patchy, non-perfused areas which were clearly outlined by surrounding, dehydrated, amber-colored glycerolized areas.

External and internal examination of the brain and spinal cord revealed surprisingly good blood washout of the central nervous system. While grossly visible infarcted areas were noted, these were relatively few and were generally no larger than 2 mm to 3 mm in diameter. With few exceptions, the pial vessels were free of blood and appeared empty of gross emboli. One striking difference which was consistently observed in FIGP animals was a far less profound reduction in brain volume during glycerolization (Figure 17). This may have been due to a number of factors: lower flow rates, higher perfusion pressures, and the increased capillary permeability and perhaps increased cellular permeability to glycerol.

Figure 16: The eye of an FGP animal following cryopreservation. The cornea has become concave due to the glycerol-induced osmotic evacuation of the aqueous humor. The vitreous humor is completely obscured by the lens which has become white and opaque as a result of the precipitation of the crystallin proteins in the lens.

Whereas edema was virtually never a problem during glycerolization of FGP animals, edema was universal in the FIGP animals after as little as 30 minutes of perfusion. In the central nervous system this edema was evidenced by a “rebound” from initial cerebral shrinkage to frank cerebral edema, with the cortices, restrained by the dura, often abutting or slightly projecting into the burr hole. Marked edema of the nictating membranes, the lung, the intestines, and the pancreas was also a uniform finding at the conclusion of cryoprotective perfusion. The development of edema in the central nervous system sometimes closely paralleled the beginning of “rebound” of ocular volume and the development of ocular turgor and frank ocular edema.

Figure 17: The appearance of the brain of an FIGP animal following cryoprotective perfusion as seen through a craniotomy performed over the right temporal lobe. The cortical surface (CS) is retracted ~3-5 mm from the cranial bone (CB) and appears

In contrast to the relatively good blood washout observed in the brain, the kidneys of FIGP animals had a very dark and mottled appearance. While some areas (an estimated 20% of the cortical surface) appeared to be blood-free, most of the organ remained blood-filled throughout perfusion. Smears of vascular fluid made from renal biopsies which were collected at the conclusion of perfusion (for tissue water determinations) revealed the presence of many free and irregularly clumped groups of crenated and normal-appearing red cells, further evidence of the incompleteness of blood washout. Microscopic examination of recirculating perfusate revealed some free, and a few clumped red cells. However, the concentration was low, and the perfusate microhematocrit was unreadable at the termination of perfusion (i.e., less than 1%).

The liver of FIGP animals appeared uniformly blood-filled throughout perfusion, and did not exhibit even the partial blood washout evidenced by the kidneys. However, despite the absence of any grossly apparent blood washout, tissue water evaluations in one FIGP animal were indicative of osmotic dehydration and thus of some perfusion.

The mesenteric, pancreatic, splanchnic, and other small abdominal vessels were largely free of blood by the conclusion of perfusion. However, blood-filled vessels were not uncommon, and examination during perfusion of mesenteric vessels performed with an ophthalmoscope at 20x magnification revealed stasis in many smaller vessels, and irregularly shaped small clots or agglutinated masses of red cells in most of the mesenteric vessels. Nevertheless, despite the presence of massive intravascular clotting, perfusion was possible, and significant amounts of tissue water appear to have been exchanged for glycerol.

One immediately apparent difference between the FGP and FIGP animals was the accumulation in the lumen of the ileum of large amounts of perfusate or perfusate ultrafiltrate by the ischemic animals. Within approximately 10 minutes of the start of reperfusion, the ileum of the ischemic animals that had been laparotomized was noticed to be accumulating fluid. By the end of perfusion, the stomach and the small and large bowel had become massively distended with perfusate. Figure 14 shows both FIGP and FGP ileum at the conclusion of glycerol perfusion. As can be clearly seen, the FIGP intestine is markedly distended. Gross examination of the gut wall was indicative of tissue-wall edema as well as intraluminal accumulation of fluid. Often by the end of perfusion, the gut had become so edematous and distended with perfusate that it was impossible to completely close the laparotomy incision. Similarly, gross examination of gastric mucosa revealed severe erosion with the mucosa being very friable and frankly hemorrhagic.

Escape of perfusate/stomach contents from the mouth (purging) which occurs during perfusion in ischemically injured human suspension patients did not occur, perhaps due to greater post-arrest competence of the gastroesophageal valve in the cat.

Oxygen consumption in the two ischemic cats in which it was measured was dramatically impacted, being only 30% to 50% of control and deteriorating throughout the course of perfusion (Figure 12).

IV. GROSS EFFECTS OF COOLING TO AND REWARMING FROM -196°C

The most striking change noted upon thawing of the animals was the presence of multiple fractures in all organ systems. As had been previously noted in human cryopreservation patients, fracturing was most pronounced in delicate, high flow organs which are poorly fiber-reinforced. An exception to this was the large arteries such as the aorta, which were heavily fractured.

Fractures were most serious in the brain, spleen, pancreas, and kidney. In these organs fractures would often completely divide or sever the organ into one or more discrete pieces. Tougher, more fiber-reinforced tissues such as myocardium, skeletal muscle, and skin were less affected by fracturing; there were fewer fractures and they were smaller and less frequently penetrated the full thickness of the organ.

Figure 18: All of the animals in the study exhibited fractures of the white matter that transected the brain between the cerebellum and the cerebral cortices. Similarly, the spinal cord was invariable severed by fractures in several locations and exhibited the appearance of a broken candle stick. The yellow box encloses a sampling area used to determine brain water content.

Figure 19: Deep fracture of the left occipital cortex. Note the absence oif fracturing in the adjacent skeletal muscle (M) observed in FGP-1. Note that the brain appears shrunken and retracted in the brainpan.

Figure 20: Appearance of the brain after removal from the brainpan. There is a massive fracture of thew right frontal=temporal cortex which penetrates the full thickness of the cerebral hemisphere to expose the right cerebral ventricle observed in FIGP-2. The cortex appears buff colored and gives the appearance of being incompletely washed out of blood.

Figure 21: Typical fracture sites in the brain (arrows and yellow shading). The olfactory cortices and the brainstem were invariably completely severed by fractures.

In both FGP and FIGP animals the brain was particularly affected by fracturing (Figures 18, 19 & 22) and it was not uncommon to find fractures in the cerebral hemispheres penetrating through to the ventricles as seen in Figure 20, or to find most of both cerebral hemispheres and the mid-brain completely severed from the cerebellum by a fracture (Figure 18). Similarly, the cerebellum was uniformly severed from the medulla at the foramen magnum as were the olfactory lobes, which were usually retained within the olfactory fossa with severing fractures having occurred at about the level of the transverse ridge. The spinal cord was invariably transversely fractured at intervals of 5 mm to 15 mm over its entire length (Figure 21). Bisecting CNS fractures were most often observed to occur transversely rather than longitudinally. In general, roughly cylindrical structures such as arteries, cerebral hemispheres, spinal cord, lungs, and so on are completely severed only by transverse fractures. Longitudinal fractures tend to be shorter in length and shallower in depth, although there were numerous exceptions to this generalization.

In ischemic animals the kidney was usually grossly fractured in one or two locations (Figure 25). By contrast, the well-perfused kidneys of the non-ischemic FGP group exhibited multiple fractures, as can be seen in Figure 24. A similar pattern was observed in other organ systems as well; the non-ischemic animals experienced greater fracturing injury than the ischemic animals, presumably as a result of the higher terminal glycerol concentrations achieved in the non-ischemic group.

Figure 23: Appearance of a fractured kidney before removal of the renal capsule. The renal capsule has only one fracture, however when the capsule is removed, the extensive fracturing of the renal cortex and medulla become evident (Figure 24, below).

Figure 24: Fractured renal cortex from FGP-1 after removal of the renal capsule. The renal cortex is extensively fractured, the renal medulla slightly less so. Note the uniform, tan/light brown color of the cortex indicating complete blood washout and the absence of red cell trapping.

Cannulae and attached stopcocks where they were externalized on the animals were also frequently fractured. In particular, the polyethylene pressure-monitoring catheters were usually fractured into many small pieces. The extensive fracture damage occurring in cannulae, stopcocks, and catheters was almost certainly a result of handling the animals after cooling to deep subzero temperatures, as this kind of fracturing was not observed in these items upon cooling to liquid nitrogen temperature (even at moderate rates). It is also possible that repeated transfer of the animals after cooling to liquid nitrogen temperature may have contributed to fracturing of tissues, although the occurrence of fractures in organs and bulk quantities of water-cryoprotectant solutions in the absence of handling is well documented in the literature (12, 13).

There were subtle post-thaw alterations in the appearance of the tissues of all three groups of animals. There was little if any fluid present in the vasculature and yet the tissues exhibited oozing and “drip” (similar to that observed in the muscle of frozen-thawed meat and seafood) when cut. This was most pronounced in the straight-frozen animal. The tissues (especially in the ischemic group) also had a somewhat pulpy texture on handling as contrasted with that of unfrozen, glycerolized tissues (i.e., those handled during pre-freezing sampling for water content). This was most in evidence by the accumulation during the course of dissection of small particles of what appeared to be tissue substance with a starchy appearance and an oily texture on gloves and instruments . This phenomenon was never observed when handling fresh tissue or glycerolized tissue prior to freezing and thawing.

There were marked differences in the color of the tissues between the three groups of animals as well. This was most pronounced in the straight-frozen control where the color of almost every organ and tissue examined had undergone change. Typically the color of tissues in the straight-frozen animal was darker, and white or translucent tissues such as the brain or mesentery were discolored with hemoglobin released from lysed red cells.

Figure 25: The (ventral) dependent and dorsal (less dependent) surfaces of the right kidney from FIGP-1. There is extensive mottling evidencing incomplete blood washout despite perfusion with many liters of CPA solution. Fracturing is much less extensive than that observed in FGP animals not subjected to prolonged periods of post-arrest ischemia. Note the pink colored “drip” from the organ that is present on sectioning board.

Figure 26: Appearance of the kidney from FIGP-1 shown above on cross-section. The renal medulla appears congested and blood filled.

The FGP and FIGP groups did not experience the profound post-thaw changes in tissue color experienced by the straight-frozen controls, although the livers and kidneys of the FIGP animals appeared very dark, even when contrasted with their pre-perfusion color as observed in those animals laparotomized for tissue water evaluation.

Figure 3-1:Fluorine and carbon; the two building blocks of the remarkable molecules knows as the perflurochemicals (PFC)s.

Physical Chemistry and Synthesis

Perfluorchemicals (PFCs) are derived from hydrocarbons by replacing hydrogen atoms with fluorine atoms, typically using common organic hydrocarbons as substrates. This is accomplished by one of three methods; the oldest of which is via a highly exothermic vapor-phase reaction employing fluorine gas. An alternative method is the more stable cobalt trifluoride technique which was developed during the Manhattan Project in World war II (WWII) [290]. Electrochemical fluorination, developed by Simmons in1950 [291] has increasingly replaced the earlier techniques.

Ectrochemical fluorination yields more homogeneous products with less carbon-carbon bond cleavage and is better suited to smaller scale production of molecules for use in research and development applications. However, whether done by electrolysis or by using the pre-Simmons method of reaction with high-valent metal fluorides, both laboratory and industrial scale PFC manufacturing and synthesis have in the past resulted in impure and poorly characterized compounds (unless perfluorinated building blocks are employed as starters). This occurs because the large difference (~15 kcal per M-1) in the carbon-hydrogen bond versus the carbon-fluorine bond energies (and the release of this energy during the synthetic process) results in undesired side reactions, isomerisations, and polymerizations creating a mélange of compounds which are not fully characterized, let alone purified.[292]

PFC carbon chain length is variable, and these, in addition to the attached moieties, determine the individual properties of a given molecule. Liquid PFCs that are useful as gas and/or heat exchange media in the lungs all exploit the utterly unique properties of the carbon-fluorine (C-F) bond and the larger size of fluorine atoms compared to hydrogen atoms. The C-F bond is the strongest covalent bond found in organic chemicals (average 485 kJ mol-1, compared to ~413 kJ mol-1 for a standard C-H bond) [293] and this bond-strength is amplified as the number of fluorine atoms on each carbon atom increases.[293]

The electroattracting nature of the fluorine atoms further increases the strength of the C-C bonds and the larger size of fluorine atoms (estimated van der Waals radius of 147 versus 120 pm) [294] and their high electron density result in a compact electron shield that ensures effective protection of the molecule’s backbone. The dense electron shell of the fluorine atoms may also provide protection against nucleophilic attack. The PFCs thus have very high intra-molecular (covalent) bonding and very low intermolecular forces (van der Waals interactions).[295]

Physical Properties

These liquids are clear, colorless, odorless, non-conducting, nonflammable, and are both hydrophobic and lipophobic. Replacement of hydrogen atoms by fluorine atoms results in high thermal stability and chemical inertness. PFCs do not undergo decomposition (except at temperatures above ~350ºC) and they are not metabolized or acted upon by any enzymatic system. They are ~1.8 times as dense as water; and are capable of dissolving large amounts of physiologically necessary gases (45 to 55 ml O2/dl and16 to 210 ml CO2/dl).[296] O2 dissolution in PFCs is via O2 occupying intermolecular sites in the liquid, unlike the pH and concentration dependent porphyrin binding in hemoglobin which is responsible for the sigmoidal oxyhemoglobin dissociation curve.[297] O2 solubility in PFCs is a linear function of the pO2 (per Henry’s law) and the structure of the particular PFC.[298] Solubility of O2 and other gases in PFCs is a function of the NMR T1 relaxation constant which determines the intermolecular cavity size and the presence and character of large channels within the liquid. Linear aliphatic structures more easily allow the formation of such channels while planar structures result in more closely interlocked layers which accommodate less gas. While the solubility of O2 is greater in aliphatic than in aromatic PFCs this is not the case with CO2. O2 solubility increases with the degree of fluorination and the O2-dissolving capacity of aliphatic PFCs having 9-11 carbons is in the range of 40 to 60 mole fractions of O2, or roughly twice that of aromatic molecules.

The O2 content of perfluocarbons at 1 atmosphere (atm) of 100% O2 is approximately 20 times that of water, twice that of blood, and 1.5 times that of an equal volume of gaseous O2. If the PFCs are compared to blood in terms of O2 carrying capacity under normal atmospheric conditions it is immediately obvious that PFCs carry only a fraction of the O2 that hemoglobin does. However, it is not simply the O2 dissolving capacity of PFCs that is important, but also their O2 delivery capability. The O2 diffusion rate from the PFC-filled alveoli to the alveolar capillaries is quite high when saturated with O2 at 760 torr [299] and due to their 50% greater O2 carrying capacity than O2 delivered as a gas at an FiO2 of 100%, the delivered O2 to the blood is 25% to 50% greater than is possible using 100% gaseous O2 for ventilation. The use of such a high FiO2 is sustainable with PFCs because, for reasons not yet understood, O2 toxicity does not occur in their presence as a liquid in the lung under normobaric conditions.

A remarkable and essential feature of the PFCs in liquid assisted ventilation is that they are essentially insoluble in both water (7 to 11 ppm at STP) and alcohols, and are only sparingly soluble in some lipids.[299] The PFCs have a kinematic viscosity (ratio of viscosity to density) similar to that of water [300] and an extremely low surface tension (15 to 19 dyn/cm2) and dielectric constant; again secondary to weak intermolecular forces resulting from the exterior fluorine atoms.[301] The volatility of PFCs varies widely depending upon the molecular weight of the molecule, with vapor pressures ranging from ~1 to over 80 torr at 25ºC. Vapor pressure is the critical determinant of the half-life of elimination of a given PFC from the lungs following both intrapulmonary and intravenous administration. Vapor pressure also dictates the viscosity of a given PFC and thus PFC-gas and PFC-surface shear interactions.[302],[303] Vapor pressure is also a critical determinant of toxicity as will be discussed under the heading Toxicology, below.

Commercially Available PFCs

Historically, the requirements of a PFC for use as a gas exchange medium in liquid ventilation are excellent solubility for respiratory and putative therapeutic gases such CO, NO, and H2S, kinematic viscosity in the range of ~ 0.50 to 2.2, low to moderate vapor pressure (1 to 15 torr) with a cutoff of 20 torr, a high spreading coefficient and a very low surface tension.[303] Additionally, a PFC that is to be used for intrapulmonary heat exchange must have a viscosity and freezing point appropriate to the application. These criteria, particularly with respect to vapor pressure and viscosity, may change in the future, depending upon the application, medical condition being treated, or large airway diameter of the patient.

No existing PFC combines all of these desirable properties, and certainly no single PFC can be tailored to have the widely varying physical properties required for a particular pathology or patient. In addition, from a physical chemistry standpoint, no single PFC is likely to combine all these properties, even in the sphere of providing assisted ventilation in ARDS; and recent research has begun to focus not only on the creation of novel of PFCs for liquid assisted ventilation applications, but also on investigating mixtures of different PFCs to provide an optimum ventilating medium which can be formulated to meet the needs of a given application.[304]

Unfortunately, de novo flurochemical synthesis involves the use of extremely toxic and hazardous materials such as fluorine gas, hydrogen fluoride, silver difluoride, or the halogen fluorides, and yields a poor ratio of desired end product to undesired (and costly to dispose of) side-products and waste.[293] Even flurochemical synthesis using per- and poly-fluroinated reagents, the ‘building block’ approach is costly, has low selectivity for many compounds and requires formidable expertise [305], although this is changing.[306]

Once the synthesis is completed, numerous and costly purification steps such as lengthily refluxing, spinning band distillation and reparative vapor phase chromatography must be undertaken, adding greatly to the cost, and making well characterized synthesis and purification of quantities sufficient for use in liquid assisted ventilation or blood substitutes a full-time effort and the province of expert chemists and dedicated facilities.[293] Historically, this has severely limited the development and commercial production of high purity, completely chemically characterized novel PFCs.

As a result, most of the initial work in liquid ventilation (including that done for liquid assisted pulmonary cooling (LAPC)) was carried out using commercially available PFCs such as perflurodecalin, Rimar-101™ (Miteni Corporation, Milan, Italy), or the Fluorinert™ liquids (3M Company, St. Paul, MN). Table 3, below, shows some of the physical properties of a number of commercially available PFCs used for leak testing, heat exchange and for cleaning applications in the electronics industry marked by 3M Corporation as the Fluorinert™ ‘liquids,’ as well as those of Rimar-101 and Perflubron™ (Alliance Pharmaceuticals, San Diego, CA).

A serious disadvantage to Fluorinert™ PFCs and all other industrial grade PFCs (as well as most reagent grade materials available from laboratory chemical suppliers) is that they are not chemically defined in terms of chain length or even precise chemical composition. As examples, FC-75 has been shown to have as many as 6 peaks when evaluated by gas chromatography and F-tripropylamine (FTPA), one of the components in the first FDA approved blood substitute, Flusol-DA, contained only 27% of perfluorinated FTPA in addition to a number of other uncharacterized compounds.[307] FC-43, a PFC used extensively as an experimental oxygen carrying blood substitute and for liquid assisted ventilation is chemically ~ 85% perfluoro-tri-n-butylamine (C12F27) with the unit structure shown in Figure 3-2. However, the perfluoro-tri-n-butylamine molecules may be present as polymers of varying lengths, and other related fluorinated molecular species are also present.

The degree of polymerization as well as the physical properties of the species which comprise the ~15% balance of FC-84, are such that the average vapor pressure, boiling point, melting point, thermal conductivity and other physical properties of FC-84 are fairly uniform from lot-to-lot.[308],[184] However, two of the most critically important determinants of the utility of PFCs in liquid ventilation applications are their vapor pressure (and thus their viscosity) and their direct chemical toxicity. Vapor pressure is of great concern, because even if the average vapor pressure of the liquid is quite low (i.e., 1.3 torr at STP for FC-43) if even a small percentage of the species present have a far higher vapor pressure, then that fraction of the liquid can turn into a gas and create long-lasting and mechanically disruptive bubbles in lung tissue under conditions of baro- and/or volu-trauma; and will create ‘sponge rubber lung’ syndrome due to stable intra-alveolar gas bubble formation by vaporizing between surfactant and the alveolar epithelium. This phenomenon, known as hyper-inflated non-collapsible lungs (HNCL) occurs even under the relatively non-traumatic conditions of PLV if the vapor pressure is low enough; as is the case with F-alkylfuran in FC-75.[309] Similarly, the unspecified and often uncharacterized other perflurocompounds (or even incompletely fluorinated compounds) may be chemically toxic to cells.[310],[311],[292]

Some Perfluorchemicals Used in Liquid Ventilation Research

Physical Property

FC-40

FC-43

FC-75

FC-

77

FC-84

PFOB

PP5/6

Rimar-101

PP50

Boiling Point (°C)

155

174

102

97

80

140.5

142

102

142

Pour Point (°C)

-57

-50

-80

-95

-95

-6

-8

-8

Vapor Pressure (torr)

3

1.3

31.5

42

79

5.2

6

31.6

6

Density (kg/m3)

1.87

1.88

1.77

1.78

1.73

1.93

1.917

1.78

1917

Coefficient of Volume Expansion (°C-1)

0.0012

0.0012

0.0014

0.0014

0.0015

0.00104

2.66

Kinematic Viscosity (cSt)

2.2

2.8

7.4

0.8

0.55

1

2.66

0.82

2.61

Absolute Viscosity (centipoise)

3.4

4.7

1.4

1.3

0.91

1

1

Specific Heat (J kg-1 °C-1)

0.25

0.25

1050

0.25

0.25

1.05

1.05

Heat of Vaporization @ B.P. (J/g)

17

17

88

20

19

78.7

78.8

Thermal Conductivity, watts (cm2) (°C/cm)

0.0006

0.0006

0.0006

0.00063

0.0006

57

0.00057

Surface Tension (dynes/cm)

16

16

15

15

13

18

17.6

15

19.3

Solubility of Water (ppm)

7

7

7

13

11

<10

<10

<10

Solubility of Air (ml gas/100 ml liquid)

27

26

25

41

43

Solubility of O2 (ml/100 ml liquid) @ 25ºC

50

52

52

50

52.7

49

52.2

49

Solubility of CO2 (ml/100 ml liquid) @ 37ºC

160

108

210

140

160

140

Molecular Weight

650

670

416.06

415

388

499

462

416.1

462

Table 3: Physical properties of some PFCs that have been used for liquid assisted ventilation: 3-M Fluorinert Liquids ™, Rimar-101, perflurodecalin and perflurooctylbromide (PFOB, Perflubron™). Perflubron™ is the first completely defined PFC intended for medical applications. Sources: [312],[292] ,[313].

Figure 3-2:Chemical structure of perfluoro-tri-n-butylamine (FC-43).

For these reasons a completely chemically defined molecule, perflurooctylbromide F3(CF2)7Br, Perflubron,™ LiquiVent™), was developed by Alliance Pharmaceuticals of San Diego, CA for use in clinical trials of liquid ventilation (Figure 25, below). Unfortunately, Perflubron™, (Figure 3-3) with a molecular weight (MW) of 498.97, has a freezing point of +6.0ºC which makes it unsuitable for use in inducing ultraprofound hypothermia (0-5oC) where the temperature of the ventilating liquid must be in the range of 2ºC to 4ºC for optimum efficacy.

Toxicology

Figure 3-3: Perflurooctylbromide (LiquiVent™)

The high stability, chemical inertness, and nearly total insolubility in both water and lipids of PFCs used in liquid assisted ventilation preclude their metabolism and limit their interaction with biomolecules. Despite 40-plus years of use in biomedicine little published work has been done on the toxicology of these compounds. Based on data from the available literature their toxicity can be divided into two categories: biophysical/biomechanical and immunological. When administered intravenously or intraperitoneally as neat (pure) chemicals injury results from the biophysical interaction with the animal rather than from biochemical interactions. Because PFCs are not miscible in water they form vascular emboli in the same way that injecting intravascular oil or air would.

PFCs with higher vapor pressures can form vascular gas emboli even if emulsified [314] and can lethally distend closed body viscuses such as the peritoneum, or cause perfluorocarbon vapor pneumothoraces (‘perflurothorax’).

PFCs of intermediate vapor pressure may accumulate in the lungs and be converted to vapor which is retained for weeks or months in the alveoli or lung parenchyma resulting in what Clark, et al., termed hyperinflated non-collapsible lungs (HNCL). [309] This phenomenon is noted at necropsy after IV administration of emulsified perflurodecalin containing blood substitutes [315] and after LAPC with intermediate vapor pressure PFCs such as FC-75 or FC-77.[272] Schutt, et al., call this ‘pulmonary alveolar gas trapping’ and they propose that the phenomenon occurs as a result of PFC liquid or vapor migration through the pulmonary surfactant-liquid bridges where it forms stable, long lasting, PFC vapor micro-bubbles. They propose that these intra-alveolar micro-bubbles are part of the ‘normal pulmonary elimination of perfluorocarbon vapor’ from the body.[316] While HNCL or ‘pulmonary alveolar gas trapping’ may not be clinically evident, and does not perturb blood gases or interfere with gas exchange, it does interfere with normal respiratory mechanics and can cause ‘stiff lungs’ in dogs following LAPC using FC-75 or FC-77.[161],[272] Stiff lungs increase the work of breathing until the vapor dissipates enough to relieve the acute tension in the alveoli.[272] As such, the author believes this phenomenon should properly be classified as an adverse biophysical effect, rather than an acceptable or normal mechanism of PFC elimination. It is interesting to note that in a chemical model of lung injury using inhaled kerosene even brief PLV with FC-77 increased mortality and resulted in extensive gas trapping.[317]

Depending upon the emulsion size intravascular PFCs may be phagocytized by PMNL’s and macrophages and be deposited in the reticuloendothelial system where they may cause enzyme induction or mild inflammation from their space occupying, mechanical effects distorting normal tissue architecture. [318] These changes are typically reversible as the PFC is eliminated via the lungs and the hepatomegaly and splenomegaly dissipate (~ 3-weeks).

The immunological effects of PFCs vary with the molecule, method of preparation and particle size (if administered intravascularly). Some PFCs appear to be directly cytotoxic to PMNLs and macrophages.[319] However, as a class, the PFCs seem to interfere with PMNL and macrophage chemotaxis, activation and de-granulation without inducing apoptosis or necrosis, by mechanisms that are not understood.[320], [321],[322] Augustin, et al., have observed that PFCs alter the cytoskeleton of hepatic macrophages in a dose dependent manner that varies with the compound. [323] Inhibition of PMNL and macrophage chemotaxis and respiratory bursts gives the PFCs moderately potent anti-inflammatory effects and by the same token makes them immunosuppressive. While this effect is immunomodulatory and probably beneficial in ARDS [324],[325],[326], it also has the potential to impair pulmonary and systemic immune surveillance and presumably increase the risk of infection and neoplasm. FC-43 has been used to delay neutrophil mediated xenograft rejection [327],[328] and Perflubron™ has been demonstrated to inhibit neutrophil activation in the rat heart after 2-hours of cold ischemia and whole blood reperfusion.[329]

A recently discovered novel and unexpected effect of at least one PFC, Perflubron™ [326], is direct inhibition of oxidative damage in both cultured pulmonary artery endothelial cells exposed to hydrogen peroxide and in linoleic acid micelles subjected to varying concentrations of the azo initiator 2,2’-diazo-bis-(2-amidinopropane) dihydrochloride . This result is unexpected because it has previously been presumed that the antioxidant activity of PFCs was secondary to their immunomodulating and immunosuppressive effects. The protective effect of Perflubron™ in a non-biological system raises many questions about its basic pharmacology, and possibly about the chemistry and environmental interactions of the PFCs as a class, should this effect prove replicable with similar compounds.

Environmental Impact and Future Availability

The PFCs may be justifiably described as the penultimate atmospheric (greenhouse) poison. The PFCs, like water vapor and methane, both absorb and emit long wave (infrared) radiation; effectively trapping heat from the sun and warming the terrestrial surface and atmosphere.

Unlike CO2 and methane, PFCs are not subject to biological cycling, are unaffected by electrochemical reactions, and do not dissociate in aqueous media. They are essentially already fully oxidized and are unaffected by standard oxidizing agents such as permanganates, chromates, and the like. As previously noted, degradation via oxidation occurs only at very high temperatures. Because of their inertness, they are similarly resistant to degradation by reduction, except under extreme conditions, requiring reducing agents such as metallic sodium. This leaves photochemical decomposition, primarily via hydroxyl radical (.OH) mediated degradation, as the only means of terrestrial disposition. Both Cicerone [330] and Yi Tang [331] have shown that the reaction of .OH with the C3 and CF4 moieties is negligible under ground-state conditions, and that the lifespan of the molecules, once they enter the atmosphere, is likely in excess of 10,000 years. The heavier, higher MW and lower vapor pressure PFCs which are ideal for liquid assisted ventilation can be expected to remain in the lower reaches of the troposphere indefinitely, and thus not reach the upper atmosphere where, however slowly, they might be photo-degraded. In any event, the PFCs are so resistant to photo-degradation that the Flourinerts, and related compounds that are used as chemically stable cooling agents in photochemical reactors, as carrier solvents for photo-decomposition of other organic molecules, and are likewise classed as ‘radiation durable compounds’ for use in the photolithography industry.[332]

At present, the PFCs constitute an insignificant contribution to greenhouse gas emissions. However, widespread medical use could change this, and in any event, 3M, DuPont and other manufactures of industrial quantities of PFCs are aggressively encouraging the use of alternative compounds which they manufacture, principally the hydrofluoroethers [333] and the perfluorinated alkyl vinyl ethers. In 1982 Riess and Le Blanc estimated that if PFC-based blood substitutes came into wide use the quantities required would be in the ‘multi-thousand-tons-per-year range’ [292] all of which would end up in the atmosphere. Widespread use of PFC-facilitated LAPC and PLV could easily require a similar amount of product.

Extensive medical use of PFCs would seem to mandate associated efforts at recovery and recycling to minimize environmental contamination. However, this is not easy to do even in hospital under controlled conditions. Recovery of PFCs used emergently for LAPC (i.e., in-field induction of hypothermia in stroke, myocardial infarction, cardiac arrest) and as the O2 carrying molecules in blood substitutes would seem to preclude effective recovery. The indefinite lifespan of these compounds makes their use akin to radiation exposure wherein the effect is cumulatively damaging, and ultimately lethal to the biosphere (as it exists now) as a consequence of their greenhouse effects and indefinite atmospheric lifespan.

The PFCs used in liquid assisted ventilation do not seem likely to accumulate or concentrate in biota. However, they do have comparatively long dwell times in patients when used clinically, and the exposure of health care workers to these volatile compounds would seem unavoidable. In light of these facts and the recent discovery that PFCs have direct radical quenching effects (with possible important environmental ramifications), as well as immunosuppressive properties, it seems reasonable to question the future large scale production, and thus the biomedical availability of these molecules.

As was the case with the first great rationalization of surgery and wound management by Pare’ [352], the creation of scientific nursing by Nightingale [353], and the development of fluid resuscitation and the first effective medical management of shock by Cannon [354] and Blalock [355], the impetus for the development of liquid ventilation was also initially warfare. Interest in the use of liquid as a breathing medium in mammals originated in the early 1960s in response to the U.S. Navy’s need to develop rescue systems for submariners that would allow them to transiently breathe saline or some other aqueous liquid. Mortality among submariners in World War I (WWI) and WW II was greater than in any other branch of military service. In WWII 22% of U.S. and 75% German submariners were killed in action.[356] With the advent of nuclear submarines in 1951, and the use of submarines to carry and deliver nuclear weapons, prolonged and complex missions while continuously submerged created the need (still largely unmet) to carry out rescue of submariners, and recovery of nuclear weapons and other strategically critical materiel, from extreme depths.

Johannes Arnold Klystra, M.D. is the Dutch pulmonologist and clinical researcher responsible for developing saline lavage of the lung as a treatment for advanced cystic fibrosis in 1958.[357] Klysta’s interests extended well beyond clinical innovation in the management of lung diseases, and in the late 1950s this maverick physician approached the Dutch Navy to explore possible ways to allow deep ocean recovery of submariners as well as the development of liquid breathing systems that would allow divers to be free from the constraints imposed by gas breathing under conditions of high pressures (Figure 4-1):

“Man has tried for centuries to invade the oceans, perhaps driven by a subconscious nostalgia for atavistic weightlessness in the vast hydrosphere that covers more than 70 per cent of the earth, but gas in his lungs, compressed by a layer of water above, confines his activities to the shallow. Nitrogen, for instance, produces a progressively severe intoxication at depths greater than100 feet and usually incapacitates a diver by ‘rapture of the deep’ at no more than300 feet. Moreover, relatively large amounts of carrier gas dissolve in blood and tissues to be released as bubbles whenever the diver returns to the surface too rapidly. These hazards are all due to the compressibility of gases. The properties of water, on the other hand, hardly change at all with pressure, and I have observed mice with fluid filled airspaces move around in no apparent distress at a simulated depth of 3000 feet. If man were able to breathe oxygenated water instead of an oxygenated carrier gas, exploration of the oceans would no longer be limited by gas toxicity and decompression sickness.”[358]

The first mammal to survive liquid breathing was a mongrel dog named ‘Snibby.’ [158] Snibby was shaved, bathed, anesthetized, intubated, and submerged in a tub of buffered salt solution in a large hyperbaric chamber at 5 atmospheres of pressure while O2 was bubbled through the saline bath. The dog breathed the liquid, which was held at a temperature of 32ºC, for 24 minutes. As Klystra noted in his published account of the experiment: “Snibby’s recovery was uneventful and he was adopted by the officers and crew of H. M. Cerberus to serve as a mascot aboard this submarine rescue vessel of the Royal Netherlands Navy.”[359]

In 1962 Klystra documented survival of mice breathing a balanced, buffered salt solution under 8 atmospheres of pressure at 20ºC for 18-hours.[158] Throughout the 1960s Klystra and his associates probed the limits of liquid breathing using aqueous solutions and they were the first to document the problem of profound hypercarbia as a fundamental limitation in tidal liquid breathing.[157] Klystra was also the first to demonstrate survival of mammals following extreme hyperbaria using spontaneous liquid breathing of a buffered salt solution.[357] A further testimony to the highly creative and innovative nature of Klystra’s work was his use of the selectively liquid lavaged lung lobe as a possible replacement for the kidney; in other words, as a mass exchanger for nitrogenous wastes and as an osmotically driven ultrafilter for removal of excess water in the setting of renal failure.[360]

One of the most remarkable things about this pioneering work is that saline and other aqueous solutions denude the alveoli of surfactant, the stiff molecular cage that supports the 3-dimensional alveolar structure and keeps the acinar airways open to ventilation. Removal of surfactant is a primary cause of serious pulmonary injury and a major pathophysiological mechanism in both acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS). Through the present, saline lavage of the lungs remains a standard model for inducing pulmonary injury to simulate ALI and ARDS in experimental animals.[361],[362] The inadequate gas carrying capacity of aqueous solutions under normobaric conditions, and the inherently injurious nature of water-based ventilating media made clinical or undersea application of liquid breathing infeasible. Indeed, breathing of balanced salt solutions, even under hyperbaric conditions in the presence of 100% O2 was only possible for extended periods of time if the animals were hypothermic. While O2 delivery was adequate, CO2 elimination was not, and hypercarbia and respiratory acidosis were lethal complications of liquid breathing under normothermic conditions.[363]

Shortly after the publication of Klystra’s pioneering work Leland C. Clark began looking for more suitable liquid breathing media. Cark’s initial efforts focused on using polyunsaturated vegetable oils such as corn and safflower oil. These oils proved injurious to lungs and Clark next evaluated the organosilanes octamethyltrisiloxane, dodecamethylpentasiloxane, decamethyltetrasiloxane, polydimethylcyclosiloxane, and polydimethylsiloxane. These compounds, commonly referred to as ‘silicone oils,’ were manufactured by the Dow Chemical Co. of Midland, MI in various chain lengths, and thus vapor pressures and physiochemical properties.[364] The organosilanes are made from a Si-O backbone to which a variety of organic groups are attached to the silicon atoms via a Si-C bond. Polydimethylsiloxane is the most common of the commercially produced organosilanes and is a polymer with a backbone consisting of a repetition of the (CH3)2SiO unit (see Figure 4-2, above). The organosilanes proved less toxic than vegetable oils, and better able to dissolve O2 and CO2, but were still too toxic to be used for liquid ventilation.

The problem of an inert and non-injurious breathing medium capable of carrying enough dissolved O2 to support life under normobaric conditions was finally solved by Leland C. Clark and Frank Gollin in 1966 with their report that a variety of fluorocarbons performed well as liquid breathing media without apparent injury to the lungs and with long-term survival of the animals.[163]

The PFC identified by Clark and Gollin, a ~50/50 mixture of isomers of F-alkylfurans (FC75), was evaluated under conditions of spontaneous respiration with the animals submerged in the liquid.[309] As proved the case with aqueous solutions, the PFCs delivered adequate amounts of O2, but failed to allow for effective clearance of CO2 resulting in lethal hypercarbia and acidosis. The vastly greater density and viscosity of PFCs relative to air or other gases limited the diffusion of CO2 into the liquid. An added problem contributing to the hypercarbia observed in liquid ventilation was the vastly greater work of breathing (WOB) imposed by a liquid 1,000 times as dense as air and the increased time for exhalation in the absence of active (negative pressure) pumping of the PFC from the lungs.

Figure 4-4: Archetypical tidal liquid ventilation (TLV) system. PFC completely replaces gas in the lungs and is moved in and out of the lungs using mechanical (usually roller) pumps. After exhalation the PFC is passed through a filter, and then through an oxygenator-heat exchanger to be scrubbed of CO2, oxygenated and warmed to body temperature before being re-infused into the lungs.

In 1970 Gordon D. Moskowitz and Thomas H. Shaffer (Figure 4-5) began work on a mechanical liquid ventilator (Figure 4-4) to overcome the problem of the increased inspiratory workload accompanying liquid breathing.[365] During the next 5-years these investigators developed progressively more sophisticated demand-regulated liquid ventilators which also began to address the need for assisted exhalation. [366],[367],[368] During the 1980s development of a variety of tidal liquid ventilators was undertaken by Shaffer, et al., and applied to animals ranging from cats [300] to preterm and neonatal lambs [369], culminating in the first clinical application of tidal liquid ventilation (TLV) to human neonates in 1989.[370]

From the beginning of liquid ventilation research with the work of Klystra in 1960, and continuing until the publication of the work of Furhman, et al. in 1991, only one kind of liquid ventilation existed; tidal or total liquid ventilation (TLV). As early as 1976 Shaffer, et al., had noticed that peak airway pressures were dramatically improved in pre-term lambs after they were returned to gas ventilation following 20 minutes of TLV.[368] The observation that lung mechanics and gas exchange remained transiently improved following TLV was extended by Shaffer, et al., in 1983 with the observations that pulmonary compliance and paO2 were increased and paCO2 was decreased during conventional gas ventilation following TLV to values lower than those that could be achieved during TLV.[300]

Bradley Furhman, an anesthesiologist and critical care physician at the University of Pittsburgh Medical School, noticed the enduring salutary effects of PLV following reinstitution of conventional gas ventilation in a model of infant respiratory distress syndrome (IRDS) using pre-term lambs, and he further noted that Shaffer, et al., had reported that these improvements in lung function did not occur following TLV in the healthy lung.[371] Furhman hypothesized that these salutary effects of TLV might be due to possible surfactant-like and alveolar recruitment effects of the comparatively large amounts of PFC retained in the lungs following TLV, since it was well documented that even with aggressive efforts to remove PFC following TLV, a volume approximately equal to the animal’s functional residual capacity (FRC) of 30 ml/kg remained in the lungs until it was eventually eliminated by evaporation.[370]

Figure 4-6: When filled to Functional Residual Capacity (FRC) the PFC forms a meniscus in the endotracheal tube. As shown at left, FRC constitutes all the volume in the lungs and trachea at the end of exhalation. (Modified by the author from the original art at Wikimedia Commons: http://en.wikipedia.org/wiki/Image:3DScience_respiratory_labeled.jpg.)

Furhman, et al., tested this hypothesis by administering FC-77 (a perfluorinated butyl-tetrahydrofuran isomer mixture) via the endotracheal tube in a volume equal to the FRC of neonatal swine.[372] As opposed to using TLV, conventional positive PPV was continued using the same parameters employed before the PFC was instilled into the lungs (Figure 4-6). This technique, christened partial liquid ventilation (PLV), proved as effective, or more effective, than TLV in decreasing airway resistance, as well as peak and mean airway pressures. PLV also seemingly abolished the need for PEEP in healthy lungs, while providing adequate gas exchange. This study not only established the effectiveness of PLV, it also posited (correctly) the mechanisms by which PLV was achieving restoration of gas exchange, increasing pulmonary compliance, and homogenizing ventilation in the lungs thus greatly reducing volutrauma (Figure 4-7, below).

Figure 4-7: Lungs from two rabbits subjected to a model of ischemia-reperfusion injury. The lung on the left (A) shows severe volutrauma to the upper lobe (‘baby lung’) with obvious consolidation in the ventral, dependent areas of the lung. The lung on the right (B) is from an animal treated with PLV and shows no evidence of injury and is homogenously ventilated with PFC and gas. Note that the letter A has been placed on an apical ‘baby lung.’

The investigators noted that due to its higher density than water the PFC rapidly flowed to the most dependent areas of the lungs and in so doing it filled collapsed alveoli and displaced serous transudate in flooded alveoli (Figure 4-9, below). With each gas breath, liquid from the large and medium caliber airways was admixed with ventilating O2 under conditions of turbulent flow, thus oxygenating it and washing it of CO2. During exhalation some, but not all of the PFC in the alveoli, flowed out into the larger airways where it was also admixed with both ventilating gas and already oxygenated PFC. During the next inspiration the alveoli were refilled with PFC that was oxygenated and cleansed of CO2. Because PFC is retained in the lungs to FRC, and gas is present in the alveoli only as micro-bubbles, if at all, the alveoli never completely empty and thus are vastly more compliant to re-inflation. As Furhman, et al., noted, the alveoli of the lungs appear to be ventilated almost exclusively with PFC throughout the ventilatory cycle with direct gas admixing occurring mostly in the larger airways (trachea, bronchi and bronchioles). Gas exchange between the blood and PFC most likely occurs due to direct PFC-alveolar membrane contact.

Also of great importance is that PLV is simple to implement; it does not require novel, complex tidal liquid ventilators with an oxygenator, heat exchanger, water trap and filters – all under complex computer control. Because there is no bulk movement of PFC over long distances of airways, the resistance to PFC flow is greatly reduced, effectively eliminating the constraint of only 5 to 7 breaths per minute in TLV.[374] Because the transit times and distances between the alveoli and the bronchioles are very small in PLV (where ventilation gas admixing and gas exchange is occurring) and because CO2 is probably exchanged in micro-bubbles in the PFC which are far smaller than would be the case in a ‘sphere’ of PFC the diameter of the alveolus (~250µ), the problem of hypercarbia is also eliminated.[375]

Figure 4-9: A: Alveoli are flooded alveoli in ARDS or pulmonary edema. When PFC is, literally, poured down the endotracheal tube it flows under gravity (due to its ~1.8x density of water) to the most dependent areas of the lungs. B: In so doing it opens the collapsed alveoli and displaces edema fluid from them. (Modified from original art by Patrick J. Lynch, medical illustrator, and is from Wikimedia Commons.)

From 1991 to 2000, PLV was extensively investigated in a number of different animals employing a variety of models of lung injury; saline FTLV, oleic acid injury, smoke inhalation, prematurity, intestinal ischemia, lung transplantation injury, and pneumococcal pneumonia.[376], [377], [378],[379],[380],[381] These studies, with no notable exceptions, showed marked benefit for PLV in ALI and ARDS.

In 1993 Leach, et al., [164] carried out the first clinical trial of PLV in IRDS (Figure 4-10). This was followed by a number of clinical trials for ARDS in both children [382] and adults.[383] These trials were sponsored by Alliance Pharmaceuticals, Inc. of San Diego, CA (Alliance) in an effort to obtain FDA approval for the use of Perflubron™ as the first gas exchange PFC in PLV.

In 2006, after 5 years of delay, the ‘definitive,’ Phase III, prospective, randomized clinical trial (RCT) of PLV in ARDS was published (LiquiVent™ study).[384] For reasons that are only now being understood, this trial showed PLV (using Perflubron™ at a dose equal to FRC (30 ml/kg), and at a lower dose of 10 ml/kg, to yield a worse outcome than conventional PPV with increased overall mortality and increased days requiring mechanical ventilation:The 28-day mortality in the control group was 15%, versus 26.3% in the low-dose (p=0.06) and 19.1% in the high-dose (p = 0.39) PLV groups. There were more ventilator-free days in the control group (13.0 ± 9.3) compared with both the low-dose (7.4 ± 8.5; p=0.001) and high-dose (9.9 ± 9.1; p =0.043) groups. Most remarkably, there was a high incidence of barotrauma: 34% pneumothoraces in the phase II LiquiVent™ trial (20% in the control group) and 29% and 28% in the phase III LiquiVent™ trial (control, 9%). Pneumothoraces requiring the placement of chest tubes is an ominous complication of PPV in ALI and ARDS and is associated with increased mortality, duration of ventilator time and length of stay in the ICU. In view of the consistently diverse, positive and well conducted animal studies demonstrating unequivocal benefit, this result was surprising.

Figure 4-10: Initiation of PLV in a neonate with IRDS. The only novel piece of equipment used was a luer-loc one-valve interposed between the endotracheal tube and the 16 mm connector to the ventilator to facilitate intra-tracheal administration of Perflubron™ without having to disconnect the patient from the breathing circuit. (Photo courtesy of Alliance Pharmaceutical, Inc.)

The reasons for the failure of the Phase III LiquiVent ™ RCT are both complicated and subtle – and are still being debated today. [385] The likely reasons for the failure of the Phase III study have important implications, not only for the future of PLV in ALI and ARDS, but also for the optimum use of PLV and LAPC in emergency and critical care medicine. The reasons for PLV’s failure are rooted in difficulties and errors that have plagued translational research from animals to humans in many areas of medical research.[386] What follows is a point-by-point evaluation of the possible causes of failure of the Phase III PLV trial as well as an analysis of the implications of these problems for LAPC.

Unanticipated Effect of Lung Protective Ventilation Strategies

The Phase III trial was designed in 1997, begun in 1998, and completed in 2002. This was well before the first report of the efficacy of lung-protective ventilation in reducing mortality in ARDS and ALI was reported in 2000 [387] and 7 years before the first influential ARDSnet study was published.[388] The criticality of minimizing barotrauma and volutrauma, even over maintaining gas exchange at optimum physiological levels, was thus not taken into consideration in the LiquiVent™ study design. This was especially significant because, due to subtle shortcomings in the design of most of the animal studies, it was not understood that PLV is a source of barotrauma and volutrauma; even when far less than full FRC-dosing is used under circumstances most like those encountered clinically (see ‘Failure to Establish a Dose-Response Curve,’ below).

While the LiquiVent™ study experimental group had a worse outcome than the control group, it should be noted that the absolute results were actually no better (or worse) than those reported in the previously cited ARDSnet studies validating lung protective ventilation (i.e., 15 to 26%). This is especially worth noting because all 3 groups of the LiquiVent™ study patients were considerably sicker than the patients in the ARDSnet studies. The objective entry criteria for the LiquiVent™ study were an initial PaO2/FiO2 <200mmHg followed by a failed (PaO2/FiO2 < 300 mmHg) response to a PEEP of ≥ 13 cm H2O at a FiO2 ≥ 0.5. By contrast, the ARDSnet patients only needed to have a PaO2/FiO2 of < 300 mmHg with no requirements for PEEP or FiO2 upon randomization. One possible reason for the superior outcome in the LiquiVent™ control group, compared to that seen in most other studies of ARDS at that time, is that Alliance selected only the very best centers of excellence in the management of ALI and ARDS to conduct the trials. By contrast, ARDSnet studies were also conducted on a contract basis by NIH at institutions that were more representative of the actual quality of care available at large metropolitan hospitals in the U.S. Alliance thus placed LiquiVent™, and consequently the entire field of PLV, on trial in a setting with the sickest patients receiving the best medical management for ARDS

Defective Translational Research Models

The animal models of ALI and ARDS used to evaluate PLV did not model the real-world course of lung injury in clinical illness. Researchers typically inflict an insult and then wait a uniform, and often unrealistically short time, for the injury to develop. By the time human patients in respiratory distress enter the ICU they have usually been ill for many hours, or even days, and as a consequence the degree of pulmonary compromise, and in particular, pulmonary edema, may be greater. Furthermore, animal models of lung injury are inherently more homogenous than is usually the case in human ARDS. Heterogeneity of injury is one of the hallmarks of both ALI and ARDS in humans, and heterogeneity means that normal or minimally injured areas of lung will be subjected to the same conditions as more severely injured areas (see discussion of varying requirements for PEEP depending upon the degree of injury individual alveoli under the heading, ‘The PFC Air Interface and Shear Effects in the Small Airways,’ below).

These observations have other important implications because loading to the theoretical FRC (30 ml/kg) in ill and often aged humans may not be possible, in the sense that the ‘normal’ or predicted volume of lung to be recruited may not be available. Alveoli can be filled with PFC only if there is enough room in the thorax to allow them to fill. If fluid in severely edematous lung parenchyma stubbornly resists relocation to the vascular compartment due to hypoalbuminemia and an interstitial pressure in excess of the hydrostatic force generated by the PFC, or if for other mechanical reasons there is not sufficient volume to accommodate a FRC-dose of PFC, then the result will be volutrauma and barotrauma to the less dependent lung. This possibility was noted by Cox, et al., as early as 1997 and was later raised in a study done by Lim et al., in 2000.[389]

Failure to Establish a Dose-Response Curve

While as previously noted, a wide range of animal models, and models of injury were investigated with respect to PLV, there was little study to determine the optimum dose-response curve of either LiquiVent™ or other PFCs used in PLV. In hindsight this seems strange because in the experimental evaluation of any novel drug the first step is usually to establish a dose-response curve and thus to bound the ‘safe and effective dose.’ This was not done in PLV and those studies which documented injury from PLV in normal lungs at FRC dosing were arguably not given the attention they deserved.[390],[391]

Recently, the work of Dreyfuss and Ricard [392] has demonstrated that dosing to FRC in healthy rats actually causes alveolar capillary leak and induces lung injury. In a series of elegant studies they have examined the complex relationship between PFC dose, PEEP and Pplat. Their studies indicate that the probable ideal dose of PFC (or of Perflubron™ in this case) is ~ 3 ml/kg; 10% of FRC, and still only 30% of the 10 ml/kg dose used in the ‘low dose’ LiquiVent™ study.[393] Furthermore, these investigators have documented that FRC dosing with Perflubron™ causes gas trapping in the lungs, and that under these circumstances, paradoxically, PEEP is protective.[391]

Gas Trapping and Selection of the Appropriate PFC

Perflubron’s™comparativelylow vapor pressure is similar to that of perflurodecalin and thus probably results in a lower spreading coefficient relative to most other PFCs used in the animal studies. This would likely result in slower dynamic flow during inspiration and in ‘plugging’ of medium caliber airways (with the previously noted effect of gas-trapping) due to high gas-fluid interfacial tension.[394],[395] These effects may contribute to barotrauma and volutrauma when Perflubron™ is administered to full FRC.[396],[397]

It now appears that if PLV is to have any chance at conventional clinical application in the West, clinical trials will have to start from scratch, quite possibly with a different PFC, or blend of PFCs, being used to achieve the pharmaco-physical properties required for the particular application – including possibly tailoring the medium to the size of the patient’s intermediate sized airways (which are greatly different between neonates, children and adults) and to the particular application at hand. For instance, as Jeng, et al., (from Schaffer’s group) states:

“In terms of clinical application, the appropriate fluid for liquid assisted ventilation will depend on the clinical situation. For example, a more viscous fluid may be more appropriate for supporting the lung during extracorporeal membrane oxygenation during which the PFC application is aimed at preventing atelectasis and fluid flux across lung-at-rest conditions. In contrast, a less viscous fluid may be preferred for TLV during which tidal volumes of fluid are exchanged. For PLV, a fluid with low vapor pressure would reduce dosing requirements during the course of the treatment. Thus, the data presented herein further relate fluid physical properties with liquid ventilation applications.”[304]

The PFC Air Interface and Shear Effects in Small Airways

Figure 4-11:The shear- inducing effect of a single flooded alveolus on neighboring alveoli. Open alveoli (A) expand evenly in unison and experience no shear. After alveolar flooding and collapse (B) shear forces occur on the adjoining alveolar septa. PFC filled; partially filled and unfilled alveoli and other acinar structures will be subject to the same damaging shear forces as is the case when aqueous media are present in the acini.

Although the alveolar air-liquid interface is eliminated during PLV, giving PLV its PEEP and surfactant-like properties, a PFC-gas interface is created. The law of Laplace ( P = 2γ/r) describes the relationship between the pressure to stabilize an alveolus (P) and surface tension at the gas-PFC interface of an alveolus (γ) in relation to the radius of the alveolus r. [398] In the normal lung, surfactant present at the gas-liquid interface lowers the air-liquid surface tension in lockstep with decreasing alveolar radius (to nearly 0 mN · m-1 for low alveolar radii), thus keeping the ratio of γ/r of the alveolus constant and guaranteeing alveolar end expiratory stability at low pressures.[399],[400] By contrast, PFCs exhibit a constant gas-PFC surface tension for any alveolar radius. This means that at the end of exhalation, the alveolar radius will be quite small, while the surface tension remains unchanged, and therefore very high compared to when the alveolus is inflated. Thus, the alveoli will collapse unless they are supported by PEEP from the ventilating gas.[401] The implication is that PFC-derived ‘liquid PEEP’ must always be balanced by precisely the right amount of ‘gas PEEP’ to prevent end-expiratory collapse of non-PFC-filled alveoli (Figure 4-11). This presents a formidable challenge in both the laboratory and the clinic.

Partial Liquid Ventilation and the Law of Laplace

Figure 4-12:In attempting to determine the extent to which PFC is likely to cause alveolar injury due to gas-liquid mediated shear forces, and variable distention of alveoli filled with PFC as opposed to gas, it is instructive to compare the dynamic behavior of PFC (red line) with surfactant (green) as well as with other liquids, such as pulmonary edema transudate (yellow) (which contains dissolved surfactant), and saline (blue line). While PFC exerts far less surface tension than saline, it still exerts a force at the air-PFC interface of ~20 dynes cm-1, and, like saline, lacks the dynamic responsiveness to changes in surface area which are the unique property of surfactant and surfactant containing solutions.

In addition to the problem of end-tidal alveolar collapse, high shear forces at the alveolar membrane as a result of insufficient PEEP during PLV may cause alveolar rupture and consequently gas/PFC leak and the development of pneumo- and/or perflurothorces.[402] The complexity and difficulty of this problem becomes clearer when consideration is given to the fact that the amount of ventilator-applied ‘gas PEEP’ will be a function of the pathological condition of each alveolus (which will vary widely in the same lung, let alone from patient to patient). This is so because the presence of a thin film of PFC in the alveolus will only be beneficial in alveoli where the native surfactant has failed to maintain alveolar patency (diameter).

In those compromised alveoli that are unable to reduce surface tension to the gas-PFC interfacial tension, the level of ‘gas PEEP’ required to keep them open at the end of exhalation will be lower during PLV than the level of ‘gas PEEP’ during conventional mechanical ventilation. Conversely, in alveoli with functioning surfactant, (i.e., able to reduce their surface tension to below that of the gas-PFC interface) there will be an interaction between PFC and the normal alveolar membrane fluid, leading to levels of ‘gas PEEP’ with PLV higher than those required to keep the alveoli open with ‘gas PEEP’ required in conventional ventilation alone (Figure 4-12). In the clinical milieu this implies careful titration of PFC dose during initiation and maintenance of PLV and equally careful titration of PFC ‘removal’ (i.e. evaporation) or weaning from PFC during the transition from PLV to gas-only PPV.[401]

Even assuming that the means can be developed to determine and control these parameters, the seemingly intractable problem of inhomogeneous gas distribution during tidal gas ventilation in PLV remains. Gas will go preferentially to the least PFC liquid loaded and least dependent airways and this will likely produce over-distension and volutrauma much as happens in the edematous consolidated lung. At a minimum it will be necessary combine fluid PEEP with pressure-controlled ventilation in a way such that the pressure in any alveolus does not exceed the pressure of the ventilating gas.[381] Failure to prevent shear or alveolar hyperinflation will result not only in direct mechanical injury to the alveolar membrane and pulmonary capillaries, [403] but also in both local and systemic injury from pro-inflammatory cytokines whose release by alveolar epithelial cells is triggered by even modest shear stress or over-distension.[404],[405]

An additional source of shear injury in PLV and thus presumably LAPC is only now beginning to emerge. Research on VLI has predominately focused on the role of high inflation pressures and large tidal volumes.[404],[406],[407] However, ventilation at low lung volumes and pressures results in a different type of lung injury, in which airway instability leads to repetitive collapse and reopening of the terminal airways.[408] This type of injury is relevant to LAPC and PLV because the air-PFC interface behaves similarly to the cyclically re-inflated collapsed airway. During reopening of collapsed airways a finger of air moves through the airway generating stresses on airway walls and injuring the airway epithelium.[409],[410],[411],[412] This does not occur during normal tidal ventilation because pulmonary surfactant stabilizes the airways and prevents their collapse during exhalation.

Figure 4-13:The power of surface tension is perhaps most easily illustrated by meniscus formation at tripartite air-cylinder-liquid interface. When waterwets a small diameter tube the liquid surface inside the tube forms a concave meniscus, which is a virtually spherical surface having the same radius, r, as the inside of the tube. The tube experiences a downward force of magnitude 2πrdσ.The gas-liquid interface under the influence of the tidal forces of ventilation generates enormous shear stress on the respiratory epithelium of small caliber airways. The ‘pull’ exerted by PFC on a capillary wall is approximately 1/5th that of saline, or ~ 0.4 πrdσ; more than enough to cause endothelial cell injury during tidal ventilation. A metal paperclip floating atop a glass of water illustrates the power of surface tension.

However, in ARDS, and where bulk liquid mixed with gas is present in the small airways (including PFC) surfactant cannot act to protect the airway epithelium against the stress field exerted on the walls of these airways as bubbles move to and fro through the liquid inside them. Bilek, et al., have investigated this phenomenon in vitro and have modelled it computationally as a semi-infinite bubble progressing through a compliantly collapsed airway, as well as a bubble progressing through a rigid tube occluded by fluid. [413] In this process, the airway walls are separated in a peeling motion as the bubble traverses the airway. This effect has been indirectly documented by experimental observation in vivo. [414],[415] Airway reopening induces large and rapid changes in normal and shear stress along the airway walls. These spatial and temporal gradients of stress exert dynamic, large, and potentially damaging stresses on the airway epithelium that do not occur under one-phase steady-flow conditions.[411], [416],[415] These forces are shown schematically in Figure 4-14. The shear stresses induced by bubble progression along both collapsed and liquid filled airways cause direct trauma due to substrate stretch-induced injuries of pulmonary epithelial cells as a result of stretching of the plasma membrane causing small tears.[417],[404] Additionally, mechanical stresses from the fluid flow may stretch the plasma membrane either directly, or as a consequence of cellular deformation.

For a low profile, predominately flat region of a cell, the non-uniformly distributed load may regionally deform the membrane. In addition, the normal-stress difference could induce transient internal flows within the cell that exert hydrodynamic stresses on the intracellular surface of the cell membrane, which might injure the membrane by the same mechanisms as extracellular stresses, and additionally be disruptive to the cytoskeleton. Bilek, et al., also describe the effects of irregular airway topology on forces generated at the air-liquid interface. They note that bulges of as little as 2 μ into the lumen of an airway result in greatly amplified shear stresses. Such bulges are commonplace in healthy airway epithelium as a result of the protrusion of epithelial cell nuclei into the airway lumen. The smoothness of the pulmonary epithelium is greatly compromised in ARDS and pulmonary edema and this may be expected to exacerbate topologically mediated shear injury to the small airways. It is interesting to note that Bilek, et al., found that the addition of surfactant to their model systems abolished shear injury in both reopening and bubble- traversing, saline occluded models; perhaps as a result of moderating liquid film thickness over the surface of the airway epithelium thereby decreasing the flow resistance and stress amplification. To what extent this may be applicable in the setting of PLV or LAPC using PFCs is unclear, since presumably surfactant is only effective by being dissolved in the bulk liquid present in the airways (i.e., in the Bilkek, et al., model, saline).

Figure 4-14: Hypothetical stresses imparted on the epithelial cells of an airway during reopening. A: a collapsed compliant airway is forced open by a finger of air moving from left to right. A dynamic wave of stresses is imparted on the airway tissues as the bubble progresses. Circles show the cycle of stresses that an airway epithelial cell might experience during reopening. The cell far downstream is nominally stressed. As the bubble approaches, the cell is pulled up and toward the bubble. As the bubble passes, the cell is pushed away from the bubble. After the bubble has passed, the cell is pushed outward.

B: A fluid ‘occlusion’ in a rigid narrow channel is cleared by the progression of a finger of air moving from left to right. A dynamic wave of stresses is imparted on the pulmonary epithelial cells lining the channel wall. The circles show the cycle of stresses that the cells might experience during reopening. Far downstream, the cell is pushed forward and slightly out. As the bubble approaches, a sudden rise in pressure and a peak in shear stress occurs, pushing the cell forward and outward with much greater force. After the bubble has passed, the cell is pushed outward. Pressure gradients generated in the presence of an air-PFC interface can also be expected to create normal stress imbalances on the cell membrane over the length of the cell. (Illustration and accompanying text reproduced from Bilek, AK, Dee, KC, Gaver, DP, III. Mechanisms of surface-tension-induced epithelial cell damage in a model of pulmonary airway reopening. J Appl Physiol 94:770-783; 2003.)

These problems may only (if ever) be solved when applying PLV to ALI and ARDS by eliminating tidal gas ventilation and replacing it with high frequency oscillating ventilation (HFOV) which yields uniform and far lower mean airway and Pt pressures and abolishes peak pressures associated with inspiration [418]; although this modality did not prove superior to either PLV or HFOV alone in animal studies – albeit using high (FRC) doses of PFC.[419]

In the case of LAPC, the short duration of FTLV as compared to that required for the treatment of ARDS using PLV may not cause sufficient shear injury to be of clinical concern, particularly in the absence of extensive preexisting pulmonary pathology. In the event that shear injury does prove to be a problem, it may be possible to use HFOV in combination with a more or less continuous process of PFC introduction and removal at or near the level of the carina. Although, the extent to which HFOV would be effective in rapidly exchanging liquid, as opposed to gas, between the large and small airways is unknown.

The Best as the Enemy of the Good

Finally, another possible factor in the discrepancy between the human and animal trials of PLV was the very close control over the availability of Perflubron™ to investigators exerted by Alliance. In part, this tightly controlled and highly selective distribution of Perflubron™ to only a small number of carefully vetted researchers was driven by the high intrinsic cost of the molecule, and by the cultural and regulatory milieu currently present in the West. Gone are the days when pharmaceutical companies freely distributed putative new drugs for studies more or less upon request. The staggering cost of regulatory approval for a new drug, coupled with often irresponsible ‘research’ aimed at inciting the media frenzy that occurs whenever the toxicity or carcinogenicity of any novel or synthetic molecule (the so-called ‘cyclamate-effect’) is newly demonstrated (regardless of the lack of soundness of the experimental design), has understandably made drug developers cautious about to whom they entrust the evaluation of potentially multibillion dollar compounds. An unfortunate effect of this abundance of caution is that novel drugs are often protected from robust evaluation under more widely varying conditions that more closely approximated those seen in the real world.

Implications for SCA and LAPC

To a much greater degree than was the case with the LiquiVent™ trials (and PLV studies in general), the study being used to initially determine the feasibility of LAPC for SCA using a TLV-type approach conducted by the author and his colleagues [272] suffers from the same defects that plagued the Alliance PLV studies. This study was conducted on healthy dogs – not on animals that were undergoing CPR with the associated very high peak and mean airway pressures. As was the case in the LiquiVent™ studies, this work preceded the ARDSnet data demonstrating the importance of low tidal volume ventilation and lung protective strategies in general, including minimizing peak and plateau airway pressures. At the time this study was published the authors were, like the LiquiVent™ investigators, unaware of the adverse effects of loading with PFC to FRC – although we certainly observed volutrauma and barotrauma – and became very sensitive to the need for controlling peak and mean airway pressures, as well as to a nuanced shaping of the flow-pressure curve. Indeed, the problem of automating the ideal flow-pressure algorithm has reportedly remained elusive, and ventilation using the technique of LAPC we reported is still least traumatically performed by hand.[420]

Figure 4-15 (left): The first human cryopatient, Eleanor Williams, undergoing LAPC on 02 March, 2002; several 1,500 ml FTLVs of PFC chilled to ~4ºC were administered by the author using gravity delivery from a flexible 2-liter peritoneal dialysis bag (blue arrow) and then suctioned out. This patient experienced massive hemoptysis immediately after the start of CPS and before LAPC was initiated (red arrow). The patient hemorrhaged ~1,500 ml of blood in less than 5 minutes: underscoring the catastrophic nature of pulmonary bleeds in the setting of friable lungs and CPR. (Photo courtesy of the Alcor Life Extension Foundation.)

The recent insights into the reasons for the failure of PLV Phase III clinical trials suggest that to the extent it is possible to reduce the volume of gas breaths and of the FTLVs required to facilitate heat exchange (i.e., delivery of PFC to and retrieval of PFC from the lungs on a cyclical basis) this will likely reduce the severity of lung injury resulting from LAPC. The measured intrapulmonary pressure in patients undergoing TLV in the Phase III LiquiVent™ study were ~60 cmH20 and this resulted in a high incidence of air leak. Intrathoracic pressure in CPR is typically in the same range; 45 to 55 mmHg or ~61 to 75 cmH20.[253] The combination of LAPC at FRC with CPR is unknown territory and should be approached with caution; and hopefully also approached with additional studies in animal models of extended duration CPR with long term follow up of the animals.

360. Klystra, J., et al., Lavage of the lung. II. A report on “long term” effects in dogs and suggestions concerning possible modifications of the original technique in order to improve its applicability for the treatment of chronic progressive and fatal diseases of the kidney in man. Acta Physiol Pharmacol Neerl., 1959. 8: p. 326-36.

“At least three general mechanisms are now thought to contribute to the generation of the extrathoracic arterial-venous pressure gradient observed during conventional CPR in the dog. These factors are (a) various venous valving mechanisms are operating, (b) peripheral venous capacitance greater than arterial capacitance, and (c) arterial resistance to collapse greater than venous resistance.

The most easily understood of these mechanisms is that of a valving mechanism. Veins at the thoracic inlet and other veins leading from the brain appear to have anatomic valves that prevent retrograde flow of blood during increases in intrathoracic pressures (9, 10, 16, 18). The valves along the extrathoracic veins, and the one at the thoracic inlet appear to be important.

The second factor contributing to the generation of the peripheral arterial-venous pressure gradient is that venous capacitance is greater than arterial. Clearly, if the same amount of blood were to move from the intrathoracic arterial and from the intrathoracic venous systems into the extrathoracic arterial and venous systems, arterial pressure would rise more than venous pressure because of the differences in extrathoracic arterial and venous capacitance.

The third contributor to the peripheral arterial-venous pressure gradient is the difference in arterial and venous resistance to collapse. Venous structures readily collapse when inside pressures fall below surrounding pressures by even a small amount. Recently we showed that the in vivo carotid artery at the thoracic inlet exhibits considerable intrinsic resistance to collapse.

This resistance to collapse can be increased in the presence of vasoconstrictor agents (23). Resistance to collapse of the arterial vessels would allow blood flow to continue toward the brain despite surrounding intrathoracic pressures which exceed intravascular pressures. The veins would readily collapse at the exit to the high pressure region, i.e. at the thoracic inlet (1, 13).

Blood returns from the periphery to the central circulation between compression cycles. Extrathoracic venous pressure rises (20) when blood flows from arteries to veins during compression. Between compressions intrathoracic pressure falls to near atmospheric, and an extrathoracic-to intrathoracic venous pressure gradient appears, which leads to flow into the chest. Right heart and pulmonary blood flow is also diastolic, at least in part (7). With conventional CPR, fight heart compression may be a component of the mechanism for pulmonary flow.”